mRNA amplification

ABSTRACT

The present invention relates to a method for the amplification of mRNA of a sample, comprising the steps of i.) generating cDNA from polyadenylated RNA employing at least one primer hybridizing to said polyadenylated RNA and comprising a 5′ poly(C) or a 5′ poly(G) flank; ii.)(aa) if present, removing non-hybridized, surplus primer(s) and/or surplus dNTPs; ii.)(ab) 3′ tailing of said generated cDNA with a poly(G) tail when in step i.(a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i.(a) primer(s) comprising a 5′ poly(G) flank was employed; or ii.)(b) 3′ tailing of said generated cDNA with a poly(G) tail when in step i.(a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i.(a) primer(s) comprising a 5′ poly(G) flank was employed using an RNA-ligase, irrespective of the presence or absence of surplus primer(s) and/or surplus dNTPs; and iii.) amplifying the tailed cDNA with a primer hybridizing to the tail(s) generated in step ii(ab) or ii(b). Furthermore, the present invention relates to methods for the preparation of in vitro surrogate(s), for identifying expressed genes in a test sample, for identifying a drug candidate for therapy of a pathological condition and for in vitro detection of a pathological condition employing said method for amplification of mRNA. In addition, the present invention relates to the use of amplified cDNA(s) as obtained by the method of the invention in hybridization, interaction and/or enzymatic arrays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of PCT/EP01/03311 filedMar. 23, 2001, which claims priority to EP 00 10 6450.0 filed Mar. 24,2000, which are incorporated in their entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for the amplification of mRNAof a sample, comprising the steps of i.) generating cDNA frompolyadenylated RNA employing at least one primer hybridizing to saidpolyadenylated RNA and comprising a 5′ poly(C) or a 5′ poly(G) flank;ii.)(aa) if present, removing non-hybridized, surplus primer(s) and/orsurplus dNTPs; ii.)(ab) 3′ tailing of said generated cDNA with a poly(G)tail when in step i. (a) primer(s) comprising a 5′ poly(C) flank wasemployed or a poly(C) tail when in step i. (a) primer(s) comprising a 5′poly(G) flank was employed; or ii.)(b) 3′ tailing of said generated cDNAwith a poly(G) tail when in step i. (a) primer(s) comprising a 5′poly(C) flank was employed or a poly(C) tail when in step i. (a)primer(s) comprising a 5′ poly(G) flank was employed using anRNA-ligase, irrespective of the presence or absence of surplus primer(s)and/or surplus dNTPs; and iii.) amplifying the tailed cDNA with a primerhybridizing to the tail(s) generated in step ii(ab) or ii(b).Furthermore, the present invention relates to methods for thepreparation of in vitro surrogate(s), for identifying expressed genes ina test sample, for identifying a drug candidate for therapy of apathological condition and for in vitro detection of a pathologicalcondition employing said method for amplification of mRNA. In addition,the present invention relates to the use of amplified cDNA(s) asobtained by the method of the invention in hybridization, interactionand/or enzymatic arrays.

2. Description of the Related Art

Several documents are cited throughout the text of this specification.The disclosure content of each of the documents (including anymanufacturer's specifications, instructions, etc.) is herewithincorporated by reference.

The study of gene expression and gene expression patterns have latelybeen revolutionized by global analysis of mRNA expression on cDNA filterassays or cDNA micro arrays (see, inter alia, Southern, Trends Genet. 12(1996), 110–115; Debouck, Nat. Genet. 21:48–50 (1999); Hacia, Nat.Genet., 21, 42–7 (1999); Cole, Nat. Genet. 21, 38–41 (1999); Bowtell DD., Nat. Genet., 21, 25–32 (1999); Cheung, Nat. Genet., 21, 15–19(1999); Duggan, Nat. Genet., 21, 10–14 (1999); Southern, Nat. Genet.,21, 5–9 (1999)). For example, Lockhart (Nature Biotechnology 14 (1996),1675–1680) describes an approach that is based on hybridization of alarge number of mRNAs to small, high-density arrays containing tens ofthousands of synthetic oligonucleotides, allowing for the simultaneousmonitoring of tens of thousands of (expressed) genes. Further microarrays for gene expression have been described in Shalon (Pathol. Biol.46 (1998), 107–109), Lockhardt (Nuc. Acids Symp. Ser. 38 (1998), 11–12)or in Schena (Trends Biotech. 16 (1998), 301–306). However, one of themajor draw-backs of the above described cDNA-array technology is thefact that these technologies require an amount of 2.5 to 10 μg ofnucleic acid probes to be tested either in the form of mRNA, reversetranscribed RNA or amplified cDNA (see, inter alia, Schena (Science 270(1995), 467–470 and PNAS U.S.A. 93 (1996), 10614–10619) or Lockhardt(1996) loc. cit.). This amount of material is normally only derived froma large of number of cells such as about 10⁹. Bryant, PNAS U.S.A. 96(1999), 5559–5564 or Mahadevappa, Nat. Biotech. 17 (1999), 1134–1136reported such an approach using at least from 50000 cells. The smallestnumber of cells yet used for ex-vivo tissue analysis and correspondinggene expression has been 1,000 cells (Luo, Nat. Medicine 5 (1999),117–122). However, a plethora of physiological and/or pathologicalconditions would require to study the gene expression pattern or“transcriptome”, defined as the entirety of mRNA molecules in a givenbiological sample (Velculescu, Cell, 88, 243–251 (1997) of a lowernumber of cells or even a single cell. For instance, the investigationof spatially and temporally regulated gene expression in embryogenesiswould clearly profit from a method were a low number of cells, inparticular a single cell, can be deduced. Similarly, it would be of highinterest to investigate the gene expression pattern/transcriptome ofindividual cells or a low number of cells derived from adult tissue,like, inter alia, blood or neuronal (stem) cells. Furthermore, multiplepathological conditions could be clarified, e.g., the delineation ofderegulated gene expression in a typical proliferation, mutaplasia,preneoplastic lesians and/or carcinomata in situ. Other examples oflocally restricted pathological processes which could be investigatedcomprise, but are not limited to, restenosis, Alzheimer's disease,Parkinson's disease, graft-versus-host disease or inflammations inautoimmunity. Furthermore, occult micrometastasis derived from a smallcancer has dire consequences if the disseminated tumor cells survive indistant organs and grow into manifest metastases. Tumor cells left afterresection of primary tumors are currently detected in bone marrowaspirates by immunocytochemical staining with antibodies directedagainst cytokeratins (reviewed in Pantel, J. Natl. Canc. Inst. 91,1113–1124 (1999)). While several studies have established the prognosticsignificance of cytokeratin-positive micrometastatic cells in bonemarrow (Braun, N. Engl. J. Med. 342, 525–533 (2000); Pantel, J. Natl.Canc. Inst. 91, 1113–1124 (1999)), the biology of these cells haslargely remained enigmatic because of their extremely low frequency inthe range of 10⁻⁵–10⁻⁶.

The systemic spread of cancer cells requires that cells evade from thesolid tumor, distribute via blood or lymphatic vessels, crossendothelial and tissue barriers and survive ectopically as single cells.The phenotypic changes accompanying these steps are considered adevelopmental process, the so-called epithelial-mesenchymaltransformation (EMT) (Hay, Acta Anatomica, 154, 8–20, (1995);Birchmeier, Acta Anatomica, 156, 217–226 (1996)). Only a small fractionof cells disseminated from a tumor may acquire EMT-associated features(Boyer, Acta Anatomica, 156, 227–239 (1996)). The epigenetic changesleading to EMT are not known so far but may have important implicationsfor the development of future therapies.

Major technical hurdles in studying epigenetic changes of, e.g.,disseminated tumor cells or pathological modified tissue are limitedaccessibility, low frequency, unambiguous identification, and subsequenttranscriptome analysis at a single cell level or of a low number ofcells. A variety of protocols has been developed for the generation of“single cell cDNA libraries” and the global amplification of mRNA fromindividual cells (see Belyavsky, Nucl. Acid. Res., 17, 2919–2932 (1989);Brady, Methods in Enzymology, 225, 611–623 (1993); and Karrer, Proc.Natl. Acad. Sci. USA, 92, 3814–3818 (1995)). However, these procedureshave obvious drawbacks, such as the restriction to 3′-ends and aninsufficient sensitivity when PCR amplificates are hybridized to cDNAarrays.

In these procedures, variation introduced during amplification of cDNAfragments was reduced by limiting the length of the cDNAs duringreverse-transcription. This was accomplished through low substrateconditions for the reverse-transcriptase; i.e. the use of lowconcentrations of an oligo d(T) primer and low dNTP concentrations.However, there is a risk of compromising reverse-transcription andsubsequent PCR-efficiency which may lead to arbitrary results whentranscriptome/gene expression patterns of cells/single cells are to beinvestigated. Furthermore, the use of an oligo(dT) primer for PCRamplification limits the use of high annealing temperatures and thusstringent annealing conditions. Typically, annealing is performed at 42°C. (Brail, Mut. Res. Genomics 406 (1999), 45–54). As pointed outhereinabove, such an approach may be suitable for a 3′ restricted cDNAsynthesis. However, higher annealing temperatures reduce the presence ofsecondary structures in the cDNA and the likelihood of unspecificannealing to internal sequences of the cDNA, which would result inshortening of the amplificates compared to the cDNA molecules. Annealingtemperatures of the method of the invention are preferably above 45° C.,more preferably above 55° C., even more preferably above 65° C.

As mentioned hereinabove, the amount of mRNA in a low number of cells oreven a single cell is insufficient for use in direct global analysis.Therefore, global analysis of expressed mRNA (of a “transcriptome”) froma low number of cells or even an individual, single cell requiresamplification of extracted and/or reverse transcribed polyadenylatedmRNA. To date, PCR amplification of small amounts of mRNA has notresulted in reliable representation of the relative expression of mRNApresent in a certain cell/low number of cells at a specific timepoint, aspecific developmental state and/or a specific physiological state(Brail, Mut. Res. Genomics 406 (1999), 45–54), Brail (1999), (loc. cit.)conclude that the method as described by Brady (Brady (1993) (loc. cit.)is likely to introduce variation(s) in the tailing reaction or the PCRamplification steps. In particular, Brail's analysis (Brail (1999), loc.cit) showed a five-fold variation even for highly-abundant house-keepinggenes (direct comparison of GAPDH and ribosomal gene L32).

Thus, the technical problem of the present invention consists inproviding means and methods which comply with the need of a global anduniform amplification of mRNA, in particular of the transcriptome of alow number of cells or a single cell. The solution to this technicalproblem is achieved by providing the embodiments characterized in theclaims.

BRIEF SUMMARY OF INVENTION

Accordingly, present invention relates to a method for amplification ofmRNA of a sample, comprising the steps of

-   (i) generating cDNA from polyadenylated RNA employing at least one    primer hybridizing to said polyadenylated RNA and comprising a 5′    poly(C) or a 5′ poly(G) flank;-   (ii)(aa) if present, removing non-hybridized, surplus primer(s)    and/or surplus dNTPs;    -   (ab) 3′ tailing of said generated cDNA with a poly(G) tail when        in step i. (a) primer(s) comprising a 5′ poly(C) flank was/were        employed or a poly(C) tail when in step i. (a) primer(s)        comprising a 5′ poly(G) flank was/were employed; or    -   (b) 3′ tailing of said generated cDNA with a poly(G) tail when        in step i. (a) primer(s) comprising a 5′ poly(C) flank was/were        employed or a poly(C) tail when in step i. (a) primer(s)        comprising a 5′ poly(G) flank was/were employed using an        RNA-ligase, irrespective of the presence or absence of surplus        primer(s) and/or surplus dNTPs; and-   (iii) amplifying the tailed cDNA with a primer hybridizing to the    tail(s) generated in step (ii)(ab) or (ii)(b).

Polyadenylated RNA can be obtained from a sample by methods known in theart. These methods comprise oligo (dT) selection steps. The sample maybe of animal or plant origin and may be a tissue or a cell sample. Saidsample may also comprise cultured tissue(s) or cultured cell(s).Particularly preferred is a sample of human origin. Samples may beobtained by methods known in the art, which comprise, but are notlimited to atherectomy, debulking, biopsy, laser dissection ormacroscopic surgical procedures.

The here described technique and method for amplification of mRNA fromsaid sample comprises steps, wherein said polyadenylated RNA obtainedfrom a sample is employed for the generation of (a) first cDNAproduct(s) employing (a) primer(s) comprising 5′-oligo (dC)/poly (C)(-or 5′-oligo (dG)/poly (G)) flanking regions. Said 5′-oligo (dC) or5′-oligo (dG) primer preferably comprises between 8 and 20 cytosine (orguanine) nucleotides, more preferably 10 cytosine (or guanine)nucleotides, more preferably said primer(s) comprise(s) 11, even morepreferably said primer(s) comprise(s) 13, most preferably said primer(s)comprise(s) 15 cytosine (or guanine) nucleotides. It is preferred thatthe first cDNA synthesis is carried out after potentially contaminatingtRNAs or rRNAs have been removed. Such a removal can be carried out bymethods known to the skilled artisan, for example, by binding thepolyadenylated mRNA to oligo (dT)/poly(T)-coated solid supports asdefined herein and subsequent washing steps.

Furthermore, this first cDNA synthesis step comprises preferably randomprimers which are present in a concentration which is 2,000 to 8,000times higher than primer concentrations used in previous studies (forexample, of 10 nM as employed in Trumper, Blood 81 (1993), 3097–3115).It is furthermore preferred that said first cDNA synthesis, i.e. thegeneration of cDNA from polyadenylated RNA, is carried out in acorrespondingly high concentration of dNTPs, preferably in aconcentration of 0.5 mM dNTPs. This first cDNA preparation step (step“i”) may also comprise means for labeling the resulting cDNA. Labels maybe introduced by methods known to the skilled artisan (see, inter alia,“Current Protocols in Molecular Biology”, edited by Ausubel et al., JohnWiley & Sons, USA (1988)), and may comprise the employment of labeleddNTPs (like biotin-labeled, radio-labeled or fluorescein-labeled dNTPs).This first cDNA synthesis step (reverse transcription), employingpreferably randomized primers, may comprise the use of standard enzymes,preferably RNAse H deficient reverse transcriptase, like Superscript IIReverse Transcriptase (GIBCO).

Since high dNTP concentrations improve said first cDNA synthesis but mayinterfere with any subsequent reactions (like tailing reactions) it ispreferred that (before carrying out any further reactions and/or stepsof the method of the invention) free surplus dNTPs are removed. Surplus,non-hybridized primer(s) are preferably also removed before additionalsteps are carried out. Said removal can be obtained, inter alia, bywashing steps, like buffer exchanges (as shown in the appendedexamples), or by filtration methods (i.e. over size-selectivemembranes). However, said removal step can also be omitted should nosurplus of dNTPs and/or primers be present. Furthermore, the removalstep can be avoided, should the subsequent “tailing-step” be carried outby an RNA-ligase step.

The 3′-tailing reaction of the method of the present invention (see step(ii)(ab) or (ii)(b) of the method of the invention) comprises thetailing with poly(G) when in step “i” (a) primer(s) comprising a 5′poly(C) flank was/were employed or a poly(C) when in step “i” (a)primer(s) comprising a 5′ poly(G) flank was/were employed. Asdemonstrated in the appended examples, it has surprisingly been foundthat, inter alia, poly(C) primers binding to poly(G)-tails are at least100-times more sensitive than poly (T) primers binding on poly(A) tails,as proposed in the prior art (Brady (1993), loc. cit.; Trumper, Blood,81, 3097–3115 (1993)).

The tailing reaction may be carried out by employing an enzyme with 3′terminal deoxynucleotide transferase activity, preferably in anon-cacodylate containing storage buffer, like terminal deoxynucleotidetransferase (MBI Fermentas; Pharmacia) However, it should be mentionedthat said “tailing”-step can also be carried out by RNA-ligase (see:Edwards, Nucl. Acids Res., 19, 5227–5232 (1991)). In this case,oligo(dC) or oligo(dG) flanking regions may be ligated to the 3-end ofthe single-stranded cDNA molecules by said RNA ligase. Sequences of theflanking regions are capable of hybridizing to the flanking region ofthe cDNA synthesis primer(s), (Edwards, Nucl. Acid Res. 19 (1991),5227–5232).

Finally, the polyG/polyC-tailed cDNA can be further amplified sincethese cDNA(s) comprise(s) a 5′ primer-introduced oligo(C) (or -G)stretch and a 3′ oligo(G) (or —C) stretch introduced by, e.g., terminaldeoxynucleotide transferase. This second PCR reaction may be carried outin the presence of labeled nucleotides. Preferred are biotin-labeled,fluorescein-labeled, dioxygenin-labeled or radio-labeled nucleotideswhich are known in the art. Furthermore, it is within the scope of thisinvention that “tagged” oligonucleotide primers (like biotin-,fluorescein-, dioxygenin-, or radio-labeled oligonucleotide primers.)are employed in order to obtain a single tag/label per cDNA species.

In a preferred embodiment of the method of the invention, said at leastone primer in step “i” is a random primer, a oligo(dT) primer or acombination thereof. Said random primer may comprise a stretch of 4 to10 random nucleotides, preferably a stretch of 5 to 9 randomnucleotides. Most preferably said random primer comprises a randomhexamer or a random octamer oligonucleotide. It is particularlypreferred that said random primer has a sequence as shown in SEQ ID NOs:1–8. Even more particularly preferred is the random primer CFI5CN6, asemployed in the appended examples comprising the nucleotides5′-(CCC)₅GTCTAG-A(N)₆ (SEQ ID NO: 8).

As shown in the appended examples, said random primer(s) can also beemployed in combination with other random primers or (an) oligo(dT)primer(s). For example, in step “i” of the present invention a primerpair (CFI5c8, corresponding to SEQ ID NO: 9) and (CFI5cT, correspondingto SEQ ID NO: 10) may be employed, comprising the sequences5′-(CCC)₅GTCTAGA(N)₈ and 5′-(CCC)₅GTCTAGATT(TTT)₄TVN, wherein “V”represents G, C or A and N represents G, T, C or A. Therefore, it isparticularly preferred that a combination of a poly d(C)/(G) primercomprising an octamer (see, e.g. SEQ ID NO: 9) is employed incombination with an oligo (dT) primer (see, SEQ ID NO: 10).

Accordingly, in a further preferred embodiment of the method of thepresent invention, the oligo(dT) primer to be employed in step “i” hasthe sequence as shown in SEQ ID NO: 10, comprising the sequence5′-(CCC)₅GTCTAGATT(TTT)₄TVN. As mentioned, hereinabove, said oligo (dT)primer(s) to be employed in step “i” of the method of the presentinvention can be used alone or in combination with (a) random primer(s)as described hereinabove. Said oligo (dT) primer(s) is/are preferably aprimer comprising an oligo (dT) stretch.

In another preferred embodiment of the method of the present invention,the concentration of said at least one primer in step “i” is in therange of 0.01 μM to 500 μM, preferably in the range of 0.1 μM to 200 μM,more preferably in the range of 1 μM to 100 μM, even more preferably inthe range of 10 μM to 60 μM. As shown in the appended example, the mostpreferred concentration is about 50 μM.

In yet another preferred embodiment of the method of the presentinvention, said primer in step “iii” comprises a stretch of at least 10,preferably at least 12, most preferably at least 15 nucleotides capableof hybridizing with the tail(s) generated in step “ii(ab)” or “ii(b)”.It is preferred that said primer does not comprise more than 20nucleotide capable of hybridizing with the tail(s) generated in step“ii(ab)” or “ii(b)” of the method of the present invention. In apreferred embodiment said primer in step “iii” has the sequence asdepicted in SEQ ID NO: 11, 12, 13, 14 or 15. As shown in the appendedexamples a particular preferred primer in step “iii” is the primer “CP2”comprising the nucleotide sequence 5′TCAGAATTCATG(CCC)₅ (see SEQ ID NO:14), with which particularly good results have been obtained in this“global amplification” step. Therefore, should a single primer beemployed in this step, the above described “CP2”-primer is particularlypreferred when in step “ii(ab)” or “ii(b)” a poly(G)-tailing was carriedout. An advantage of employing only a single primer in step “iv” of theinvention is that potential “primer-primer” interactions can be avoidedand relatively high primer concentrations preferably above 0.2 μM, morepreferably above 0.8 μM, even more preferably above 1,0 μM can be used.Higher primer concentrations above 1,0 μM or 1,2 μM may also beemployed.

In another preferred embodiment of the method of the present invention,said polyadenylated RNA (and/or mRNA to be amplified) is bound to asolid support. Said solid support may be, inter alia, a bead, amembrane, a filter, a well, a chip or a tube. Particularly preferred isa magnetic bead, a latex bead or a colloid metal bead. However, saidpolyadenylated RNA may also be bound on solid supports like polystyrenebeads. Solid phases known in the art also comprise glass and/or siliconsurfaces, nitrocellulose strips or membranes and plastic (test) tubes.Suitable methods of immobilizing nucleic acids, in particularpolyadenylated RNA on solid phases include but are not limited to ionic,hydrophobic, covalent interactions and the like. The solid phase canretain one or more additional receptor(s) like, for example, a poly (T)stretch, which has/have the ability to attract and immobilize thepolyadenylated RNA. This receptor can also comprise a changed substancethat is oppositely charged with respect to the nucleic acid. In a mostpreferred embodiment of the method of the present invention, the solidsupport, (like said magnetic bead) comprises therefore an oligo(dT)stretch.

As shown in the appended examples, the mRNA/polyadenylated RNA to beamplified by the method of the present invention can easily be isolatedon an oligo (dT) coated solid support, like oligo (dT) coated magneticbeads.

In yet another embodiment of the present invention the mRNA to beamplified is derived from a tissue, a low number of cells or a singlecell. Said low number of cells may be in the range of 10⁶ to 2 cells.Said tissue, cells or single cell may be of plant or animal origin. Itis particularly preferred that said tissue, cells or single cell is/areof human origin. Said tissue, cells or single cell may be, furthermore,a pathological sample and/or a sample which is suspected to bepathological. Whether pathological, suspected to be pathological ornormal/healthy, said tissue, (low number of) cells or single cell may bederived from a body fluid or from solid tissue. Body fluids may compriseblood, lymphatic fluid, peritonal fluid, spinal/cerebrospinal fluid,amnionic fluid, urine or stool. Said solid tissue may be derived fromall animal/human organs or glands. Furthermore, said tissue may comprisemalignant transformations, like tumors or restenotic tissue. Therefore,said tissue, (low number of) cells, or single cells may also be fromcarcinomas, sarcomas, lymphomas, leukemias or gliomas. However, itshould be pointed out that the method of the present invention can alsobe employed on samples derived from benign tissue, normal tissue as wellas from cultured samples, like tissue and/or cell cultures. Tissues, lownumber of cells and/or single cells can be obtained by methods known inthe art, which comprise, but are not limited to biopsis, aspirations ordilutions. Samples can also be separated and obtained by FACS sorting orisolation by immunological methods or “receptor/ligand” binding methods.As shown in the appended examples, samples can also be obtained byartherectomy, e.g. helical device for artherectomy (X-sizer, Endicor)

In another preferred embodiment of the method of the present invention,said tissue, low number of cells or single cell is a chemically fixedtissue, chemically fixed low number of cells or chemically fixed cell.Said fixation may be carried out in (para)formaldehyde. Preferredconcentrations are in the range of 0.1 to 1%, most preferred is,however, a concentration of 0.1%. Said fixation is preferably carriedout for less than 30 minutes (when concentrations below 1% areemployed). Most preferably said fixation is carried out at a(para)formaldehyde concentration of 0.1% for 5 minutes.

In another preferred embodiment, the method of the present inventionfurther comprises a step “iv” wherein the generated amplified cDNA isfurther modified. Said modification may comprise the introduction ofmeans for detection, for example, the introduction of nucleotideanalogues coupled to (a) chromophore(s), (a) fluorescent dye(s), (a)radio-nucleotide(s), biotin or DIG. Labeling of amplificated cDNA can beperformed as described in the appended examples or as described, interalia, in Spirin (1999), Invest. Opthalmol. Vis. Sci. 40, 3108–3115.

Furthermore, it is preferred that the obtained amplified cDNA is boundto a solid support, as defined hereinabove.

Since standard cacodylate containing buffers (like some cDNA synthesisbuffers) may interfere with individual steps of the method of theinvention (like the “tailing reaction”) it is preferred that all orindividual steps are carried out in a non-cacodylate buffer.Particularly preferred is a phosphate buffer and most preferred is aKH₂PO₄ buffer as employed in the appended examples. Preferably saidbuffer is a buffer of low ionic strength (see Nelson, Methods inEnzymology, 68, 41–50 (1979)). Furthermore, the use of dGTP or dCTP in“tailing” reactions leads to short extension of 15–30 nucleotides, whilethe use of dATP or dTTP leads to long extensions ranging from 70 toseveral hundred nucleotides (Nelson (1979), loc. cit.; MBI Fermentas1998/1999 catalog, p. 125); Deng, Methods Enzymology, 100, 96–116,(1983)). Long poly(dA)/(dT) tails, however, result in non-homogeneouspopulations of cDNAs during amplification due to varioushybridization/annealing sites. In contrast, the method of the inventionwith its short (10–30 bases) 5′ primer and 3′tailing introducedoligo(dC) or oligo(dG) flanking regions generate homogenous populationsof amplified cDNAs, amplifying preferentially the coding regions of theoriginal cDNA molecules.

In yet a more preferred embodiment of the method of the presentinvention, the sample comprising mRNA/polyadenylated RNA to be amplifiedis derived from a cell and/or a tissue (or is a cell and/or a tissue),the genetic identity of which had been defined by comparative genomichybridization (CGH). As shown in the appended examples, a methodcomprising CGH of a single cell (SCOMP; see Klein (1999), PNAS USA 96,4494–4499)) has recently been described which allows for unambiguousidentification of a single cell. With this method it is possible toidentify, inter alia, a tumor cell and/or a cell of tumerous origin byits chromosomal aberrations. Employing the here described method formRNA amplification and combining said method with SCOMP, it is thereforepossible to isolate genomic DNA and mRNA from the same single cell.

The present invention also relates to a method for the preparation of anin vitro surrogate for (a) pathologically modified cell(s) or tissue(s)comprising the steps of:

-   (a) amplifying mRNA of said pathologically modified cell(s) or    tissue(s) according to the steps of the method described herein    above;-   (b) assessing the quantity and, optionally, biophysical    characteristics of the obtained cDNA and/or transcripts thereof,    thereby determining the gene expression pattern of said    pathologically modified cell(s) or tissue(s);-   (c) selecting an in vitro cell, the gene expression pattern of which    resembles the gene expression pattern of said pathologically    modified cell(s) or tissue(s); and-   (d) adapting the gene expression pattern of said in vitro cell to    the gene expression pattern of the pathologically modified cell or    tissue.

The term “in vitro surrogate” as used herein means (a) cell(s) or (a)cell line(s) which is capable of mimicking a pathological situation or apathological condition. Said surrogate may be useful, inter alia, inmedical, pharmacological or scientific experiments and may be employedfor drug screening purposes. In particular, such a surrogate cell/cellline may be employed to identify potential drugs and/or medicaments.Such identification may be carried out by screening libraries ofchemicals and/or biologics, and, preferably, said surrogate(s) is/areused in high throughput-screenings.

The assessment of the quantity and, optionally the biophysicalcharacteristics of the obtained cDNA and/or transcripts thereof can becarried out by methods known to the person skilled in the art and/or asdescribed herein.

The term “in vitro cell” as employed in accordance with this inventionpreferably relates to a cell which may be maintained in culture. Saidcell is preferably maintained in culture for at least 1 hour, morepreferably for at least 6 hours, more preferably for at least 12 hours,more preferably for at least one day, more preferably for at least twodays, more preferably for at least 3 days, more preferably for at leastone week, most preferably for several weeks.

It is particularly preferred that said surrogate/in vitro surrogatefaithfully reflects the transcriptome/gene expression pattern of thepathologically-modified cell or tissue. Said surrogate should closelyresemble the pathologically modified tissue or pathologically modifiedcell. It is therefore preferred that the “in vitro cell” as mentioned instep c. herein above is similar to the pathologically modifiedtissue/cell. For example, the “in vitro cell” may be derived from asimilar tissue or organ as the pathologically modified/diseased tissue.Inter alia, coronary artery smooth muscle cells can be employed as “invitro cells”, the gene expression pattern of which resembles the geneexpression pattern of restenotic tissue. Similar, liver cells (like,e.g, HepG2) may be employed to obtain a surrogate for pathologicallymodified liver tissue, cultured renal cells (like, e.g. ATCC 45505) forkidney diseased tissue, cardiomyoblasts (like, e.g., rat cardiomyocyte)for heart muscle diseased tissue, or NCI cell lines as described inRoss, Nat. Genetics 24 (2000), 227–235 for tumerous diseases, neoplasticdiseases or cancer.

Said “adaption” of step (d) as mentioned herein above is carried out inorder to adapt the gene expression pattern of the selected “in vitrocell” to a gene expression pattern which reflects more closely the geneexpression pattern of the pathologically modified tissue/cell. Inparticular, when it was found (in steps (a) and (b) of the method asdescribed herein above), that a particular transcript/expressed gene (ora group of particular transcripts/expressed genes) was downregulated incomparison to said “in vitro cell” (or a control cell), it should beattempted to upregulate the expression said transcript/expressed gene(or group of said transcripts/expressed genes) in said “in vitro cell”.Accordingly, should a specific transcript/expressed gene (or a group ofspecific transcripts/expressed genes) be upregulated in comparison tosaid “in vitro cell” (or a control cell), it should be attempted todownregulate said transcript/expressed gene (or a group thereof) in said“in vitro cell”. Particular methods, factors, compounds and/orsubstances which may be useful to adapt the gene expression pattern ofsaid in vitro cell are described herein below.

In one embodiment, it is preferred that said adaption step comprisescontacting said in vitro cell with at least one compound, factor,substance, a plurality of compounds, factors, susbtances or acombination thereof and assessing whether said contacting leads to amodified gene expression pattern/transcriptome in said in vitro cell.The assessment of the gene expression pattern may be carried out by themethod of the invention but may also comprise other analysis methodsknown in the art, like biochemical or biophysical methods. Particularlypreferred are hereby methods like proteome analysis, comprising one- ortwo dimensional (gel) electrophoresis, high-performance liquidchromatography, mass spectrography or antibody-based detection methods(blotting or array systems).

The above mentioned pathologically modified cell(s) or tissue(s) an/orin vitro cell is preferably of animal origin. Particularly preferred arehereby cell(s) or tissue(s) derived and/or obtained from primates,rodents or artiodactyles. Even more preferred are cell(s) and/ortissue(s) from humans, monkeys, pigs, cows, rats or mice.

In yet another embodiment, the method for the preparation of an in vitrosurrogate for

-   (a) pathologically modified cell(s) or tissue(s) comprises the    further steps of-   b(1). determining the gene expression pattern of (a) control cell(s)    or (a) control tissue(s); and-   b(2). determing the gene(s) which is/are differentially expressed in    said for pathologically modified cell(s) or tissue(s) and said    control cell(s) or tissue(s).

The here mentioned control cell(s) or control tissue(s) can be easilydetermined by the person skilled in the art. For example, similar tissuefrom a healthy donor may be employed. As shown, e.g., in the appendedexamples a control tissue for restenotic tissue may be media ormedia/intima of healthy coronary arteries. Furthermore, control cell(s)or control tissue(s) may be obtained during biopsis of hepatic tissue,renal tissue, prostate, cervical tissue etc.

It is particularly preferred that the gene expression pattern, i.e. the“transcriptome” of said control cell or control tissue is alsodetermined by employing the method for the amplification of mRNA of asample as described herein. Preferably, said transcriptome analysis ofsamples like the pathologically modified cell(s) or tissue(s), thecontrol cell(s) or control tissue(s) comprises the steps of

-   i. generating cDNA from polyadenylated RNA of said pathologically    modified cell or tissue, said control cell or tissue and/or said in    vitro cell employing at least one primer hybridizing to said    polyadenylated RNA and comprising a 5′ poly(C) or a 5′ poly(G)    flank;-   ii.(aa) if present, removing non-hybridized, surplus primer(s)    and/or surplus dNTPs;    -   (ab) 3′ tailing of said generated cDNA with a poly(G) tail when        in step i. (a) primer(s) comprising a 5′ poly(C) flank was/were        employed or a poly(C) tail when in step i. (a) primer(s)        comprising a 5′ poly(G) flank was/were employed; or    -   (b) 3′ tailing of said generated cDNA with a poly(G) tail when        in step i. (a) primer(s) comprising a 5′ poly(C) flank was/were        employed or a poly(C) tail when in step i. (a) primer(s)        comprising a 5′ poly(G) flank was/were employed using an        RNA-ligase, irrespective of the presence or absence of surplus        primer(s) and/or surplus dNTPs;-   iii. amplifying the tailed cDNA with a primer hybridizing to the    tail(s) generated in step ii(ab) or ii(b);-   iv. employing the amplified cDNA in (a) hybridization assays; and-   v. detecting differences and/or similarities in the gene expression    pattern of said pathologically modified cell or tissue, said control    cell or tissue and/or said in vitro cell

The embodiments as described herein above for the method of theinvention may be applied for said transcriptome analysis of saidpathologically modified cell(s) or tissue(s), control cell(s) or controltissue(s) and/or said in vitro cell.

The above described method for the preparation of an in vitro surrogatecan be, inter alia employed for restenotic tissue or for an restenoticcell. Said control cell or said control tissue(s) may be selected fromthe group consisting of smooth muscle cells, media/intima of (healthy)coronary arteries and media/intima of (healthy) peripheral arteries.

The “in vitro cell” to be accepted to the gene expression pattern of apathologically modified cell(s) or tissue(s) may be derived from primarycell culture, a secondary cell culture, a tissue culture or a cell line.Preferably, these cells and/or cell cultures are, but are not limitedto, cultured muscle cells, cultured smooth muscle cells, culturedcoronary artery smooth muscle cells, HepG2 cells, Jurkat cells, THP-1cells, Monomac-6 cells or U937-cells. Such cells are easily obtainablefrom sources known in the art, like DSMZ, Braunschweig or the ATCC, USA.Furthermore, cardiomyoblasts may be employed as “in vitro cell” foradaption to a “surrogate”.

Said adaption step (step d. of the above described method for thepreparation of an in vitro surrogate) may comprise the exposure of saidin vitro cell to physical and/or chemical change(s), wherein saidphysical change(s) may comprise temperature shifts, light changes,pressure, pH-changes, changes in ionic strength or changes in thecomposition of gas phase(s) (like O₂, N₂, CO, CO₂) and said chemicalchanges may comprise medium exchanges, medium substitutions, mediumdepletions and/or medium additions. It is particularly preferred thatsaid chemical changes comprise the exposure to compounds like growthfactors, hormones, vitamines, antibodies or fragments and/or derivativesthereof, small molecule ligands, cytokines, transcription factors,kinases, antibiotics, natural and/or non-natural receptor ligands, orcomponents of signal transduction pathways. Said adaptation step mayalso comprise co-culturing with other cells/cell lines, for exampleco-culturing with blood cells, glial cells, dendritic cells orosteoclasts. Said blood cell may comprise monocytes and T-lymphocytes.

In an even more preferred embodiment of the method for the preparationof an in vitro surrogate, said cytokine is IFN-γ (or a functionalderivative therof), said natural and/or non-natural receptor ligand is aligand for IFN-γ receptor (a and/or b chain), said transcription factoris IRF-1 or ISGF3-γ-(p48), said kinase is tyrosine kinase Pyk2, saidcomponents of signal transduction pathways is Dap-1, BAG-1, Pim-1 orIFN-γ-inducible protein 9–27, said growth factor is platelet growthfactor AA, angiotensin or fibroblast growth factor or said antibiotic israpamycin.

In this context, the term “functional derivative” of IFN-γ relates toderivatives that retain or essentially retain the biological propertiesof natural IFN-γ. Examples of such derivatives are muteins. The sameapplies, mutatis mutandis, for other components mentioned herein.

In vitro surrogate(s) as obtained by the above described methods areparticulary useful in drug screening methods and/or in toxicologicalanalysis. Such methods comprise, but are not limited to the detection ofmodified gene expression pattern after contacting said in vitrosurrogate with a test substance and/or a potential drug candidate. Suchscreening methods are well known in the art and are, inter alia,described in Scherf, Nat. Genetics 24 (2000), 236–244; Ross, Nat.Genetics 24 (2000), 227–235. High-throughput screenings are describedand/or reviewed in Sundberg, Curr. Opin. Biotechnol. 11 (2000), 47–53;Hopfinger, Curr. Opin. Biotechnol. 11 (2000), 97–103; Vidal, TrendsBiotechnol. 17 (1999), 374–381; Gonzales, Curr. Opin. Biotechnol. 9(1989), 624–631; Fernandes, Curr. Opin. Chem. Biol. 2 (1998), 597–603.

Additionally, the present invention relates to a method for identifyingdifferentially expressed genes in a test sample, wherein said methodcomprises the steps of (a) providing a test sample and a control sampleeach comprising polyadenylated RNA; (b) employing the steps of themethod for the amplification of mRNA of the present invention on saidtest and control sample; and (c) comparing the obtained amplified cDNAof said test sample with the obtained amplified cDNA of said controlsample. The test and control sample may be derived from the sameorganism but may also be derived from different organisms/individuals.Furthermore, said test sample may comprise tissue cultures or cellcultures. Furthermore, said test and/or control sample comprisespreferably the same kind of cell(s) and/or tissue(s). The comparison ofstep (c) can be carried out as, for example, shown in the appendedexamples and may involve hybridization of obtained amplified cDNA tocDNA arrays. The method for identifying differentially expressed genesmay therefore comprise the comparison of tissue, (a low number of) cellsor a single cell of distinct origin. For example, pathological andnon-pathological tissue, (low number of) cells or single cells may becompared on the transcriptome level.

The present invention also relates to a method for identifying a drugcandidate for prevention or therapy of a pathological condition or apathological disorder comprising the steps of (a) contacting a samplecomprising polyadenylated RNA with said drug candidate; (b) employingthe steps of the method for the amplification of mRNA of the presentinvention on said sample; and (c) detecting the presence, the absence,the increase or the decrease of particular expressed genes in saidsample.

The sample to be contacted with said drug candidate may be an isolatedorgan, tissue, (low number of) cells or a single cell. Said sample mayalso be a tissue or a cell culture sample. Furthermore, it is alsoenvisaged that a laboratory animal and/or a subject may be contactedwith said drug candidate and that after (or during) said contact acorresponding sample is obtained, for example, by biopsy.

Furthermore, the present invention provides for a method for in vitrodetection of a pathological condition or a susceptibility to apathological condition in a subject comprising the steps of (a)providing a sample comprising polyadenylated RNA from said subject; (b)employing the method for the amplification of mRNA of the presentinvention on said sample; and (c) detecting a pathological condition ora susceptibility to a pathological condition based on the presence, theabsence, the increase, the decrease or the amount of (a) expressedgene(s) in said sample.

The presence, absence, increase or decrease or amount can be detected,inter alia, by comparing the obtained cDNA(s) with obtained cDNA(s) froma healthy control sample. The sample(s) may be of human origin.

In addition, the present invention relates to the use of the amplifiedcDNA as obtained by the method of the invention for in vitro and/or invivo expression. Methods for in vitro and/or in vivo expression are wellknown in the art and are described, inter alia, (“Current Protocols inMolecular Biology”, edited by Ausubel et al., John Wiley & Sons, USA(1988); Schoelke, Nature Biotech., 18, 233–234 (2000)) or in“Biotechnology”; edited by Rehn and Reed, VCM Verlagsgesellschaft mbH,Weinheim, FRG, (1993). Furthermore, in vitro expression in plant cellsis described in Weissbach “Methods for Plant Molecular Biology”,Academic Press, San Diego, U.S.A. (1988). Particular preferred systemsfor in vitro expression are translation systems known in the art, likeE. coli lysates for coupled transcription/translation (Basset, J.Bacteriol.,(1983) 156, 1359–1362), wheat germ translations systems orreticulocyte lysates (Walter, Methods Enzymol., 93, 682–691 (1983);Dasnahapatra, Methods Enzymol., 217, 143–151 (1993); Hancock, MethodsEnzymol, 255, 60–65 (1995); Wilson, Methods Enzymol., 250, 79–91(1995)). Said in vitro and/or in vivo expression of said amplified cDNAcomprises transcription as well as translation events and, therefore,comprises the generation of mRNA as well as, if desired, of protein(s)and/or peptide(s). Therefore, the present invention also relates to theuse of amplified cDNA as obtained by the method of the present inventionfor the in vitro and/or in vivo preparation of mRNA transcripts.

The present invention also relates to the use of the amplified cDNA asobtained by the method of the present invention or of mRNA transcriptsas defined hereinabove and obtained by in vitro and/or in vivoexpression of the cDNA as obtained by the method of the presentinvention, in hybridization assays, and/or in interaction assays.

Preferably, said hybridizing assays are carried out under definedconditions. Most preferably, said hybridizing conditions are stringentconditions. However, the term “hybridizing” as used in accordance withthe present invention relates to stringent or non-stringenthybridization conditions. Said hybridization conditions may beestablished according to conventional protocols described, for example,in Sambrook, “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Laboratory (1989) N.Y., Ausubel, “Current Protocols in MolecularBiology”, Green Publishing Associates and Wiley Interscience, N.Y.(1989), or Higgins and Hames (eds) “Nucleic acid hybridization, apractical approach” IRL Press Oxford, Washington D.C., (1985).

In a preferred embodiment, said hybridization assay comprises thehybridization to oligonucleotide arrays, cDNA arrays, and/or PNA arrays,said interaction assay comprises the interaction with carbohydrate(s),lectin(s), ribozyme(s), protein(s), peptide(s), antibody(ies) or (a)fragment(s) thereof, and/or aptamer(s).

The above mentioned arrays are well known in the art (see, inter alia,Debouck, Nat. Genet. 21:48–50 (1999); Hacia, Nat. Genet., 21, 42–7(1999); Cole, Nat. Genet. 21, 38–41 (1999); Bowtell D D., Nat. Genet.,21, 25–32 (1999); Cheung, Nat. Genet., 21, 15–19 (1999); Duggan, Nat.Genet., 21, 10–14 (1999); Southern, Nat. Genet., 21, 5–9 (1999)). Inparticular, cDNA arrays may be obtained from Clontech, Palo Alto;Research Genetics, Huntsville and comprise cDNA microarrays, andoligonucleotide arrays may be obtained from Affymetrix, Santa Clara.cDNA arrays may be prepared, inter alia, according to the methodsdescribed in DeRisi, Nat. Genet. (1996), 14, 457–460; Lashkari, Proc.Natl. Acad. Sci. USA, 94, 13057–13062 (1997); Winzeler, Methods Enzymol.306, 3–18 (1999); or Schena (1995), loc. cit., oligonucleotide arrays,inter alia, according to Southern (1999), loc. cit.; Chee, Science, 274,610–614 (1996). The above mentioned arrays may comprise macroarrays aswell as microarrays.

As shown in the appended examples, the cDNA as obtained by the method ofthe present invention (or mRNA transcripts of said cDNA) can be employedon cDNA arrays/cDNA microarrays in order to deduce the gene expressionpattern/transcriptome of a (test) sample comprising polyadenylated RNA.

Hybridization assays as described herein above are useful, inter alia,in medical, diagnostic, pharmacological as well as in scientificsettings. As shown in the appended examples, it is possible to employDNA as obtained by the method of the present invention in order todeduce the (gene) expression pattern of pathologically modified cellsand/or tissues, e.g., tumerous (cells) tissues, restenotic tissue.

The appended examples document, inter alia, that the method of thepresent invention can be employed to deduce differentially expressedgenes in restenotic tissue. In this way 224 genes were identified thatare differentially expressed in restenosis, wherein 167 genes wereoverexpressed and 56 genes were underexpressed in comparison tocontrols. The detection of specific, differentially expressed genes orgene expression pattern(s) can, therefore, also be employed indiagnostic methods in order to define, inter alia, restenotic tissue.Furthermore, as described in the appended examples, the method of thepresent invention may be useful in the diagnosis of neoplastic diseases,cancer.

The amplified cDNA as obtained by the method of the present inventionis, therefore, particularly useful in establishing gene expressionprofiles of tissues and/or cells. Such gene expression profiles/geneexpression patterns may be particularly useful and important in drugdiscovery screens. It is particularly preferred that data obtained bysuch gene expression profiling be used in combination with drug activitypatterns (see, inter alia, Weinstein, Science 275 (1997), 343–349;Weinstein, Science 258 (1992), 447–451, van Osdol, J. Natl. Cancer Inst.86 (1994), 1853–1859 or Pauli, J. Natl. Cancer Inst. 81 (1989),1088–1092). Furthermore, it is envisaged that cDNA as obtained by themethod of the present invention and/or mRNA transcripts thereof be usedin assays wherein gene expression patterns and drug activity profilesare correlated as described in Scherf, Nat. Genetics 24 (2000), 236–244and in Ross, Nat. Genetics 24 (2000), 227–235. Further, the“transcriptome”-data obtained by the methods of the invention, asdescribed herein above, may also be correlated on the protein level, asdemonstrated in the appended examples.

The present invention also relates to the use of amplified cDNA obtainedby the method of the invention for sequence specific PCR, cDNA cloning,substractive hybridization cloning, and/or expression cloning. SpecificPCR can be used, e.g., to determine the relative amounts of transcriptswithin a given sample and between samples. The cDNA generated by thepresent invention could also be applied to subtractive hybridizationcloning to select for cDNAs specific for or absent from the sample whichis demonstrated in the appended examples (Rothstein, Methods Enzymol.225, 587–610 (1993); Diatchenko, Methods Enzymol., 303, 349–380 (1999)).

In a preferred embodiment, the adapter-primers Eco 44 I: 5′-GTA ATA CGACTC ACT ATA GGG CTC GAG CGG CTC GCC CGG GCA GG-3′ (SEQ ID NO: 31), Eco12 I:5′-AAT TCC TGC CCG-3′ (SEQ ID NO: 32), Eco 43 II: 5′-TGT AGC GTGAAG ACG ACA GAA AGG TCG CGT GGT GCG GAG GGC G-3′ (SEQ ID NO: 33) or Eco12 II: 5′-AAT TCG CCC TCC-3′ (SEQ ID NO: 34) may be employed with theabove mentioned method i.e. substractive hybridization analysis. In afurther preferred embodiment, the primers P1–30: 5′-GTA ATA CGA CTC ACTATA GGG CTC GAG CGG-3′ (SEQ ID NO: 35), P2–30: 5′-TGT AGC GTG AAG ACGACA GAA AGG TCG CGT-3′ (SEQ ID NO: 36), P1–33: 5′-GTA ATA CGA CTC ACTATA GGG CTC GAG CGG CTC-3′ (SEQ ID NO: 37), P2–33: 5′-TGT AGC GTG AAGACG ACA GAA AGG TCG CGT GGT-3′ (SEQ ID NO: 38), PN1–30: 5′-CGA CTC ACTATA GGG CTC GAG CGG CTC GCC-3′ (SEQ ID NO: 39) or PN2–30: 5′-GTG AAG ACGACA GAA AGG TCG CGT GGT GCG-3′ (SEQ ID NO: 40) may be employed whenamplifying the resulting cDNA populations which may be obtained by theabove mentioned substractive hybridization analysis.

In a more preferred embodiment primers primers P1–30: 5′-GTA ATA CGA CTCACT ATA GGG CTC GAG CGG-3′ (SEQ ID NO: 35), P2–30: 5′-TGT AGC GTG AAGACG ACA GAA AGG TCG CGT-3′ (SEQ ID NO: 36) are employed for theaforementioned method as shown in the appended examples.

The present invention also provides for a kit comprising at least oneprimer as defined herein above.

Advantageously, the kit if the present invention further comprises,besides said primer/primers, optionally, solid supports (such asmagnetic beads), enzymes, such as reverse transcriptase(s), RNA-ligaseor terminal deoxynucleotidyltransferase as well as (a) reactionbuffer(s) (like cDNA “wash buffer” or “tailing buffer”) and/or storagesolution(s). Furthermore, parts of the kit of the invention can bepackaged individually in vials or in combination in containers ormulticontainer units. The kit of the present invention may beadvantageously used for carrying out the method(s) of the invention andcould be, inter alia, employed in a variety of applications referred toabove, e.g., in diagnostic kits or as research tools. Additionally, thekit of the invention may contain means for detection suitable forscientific and/or diagnostic purposes. The manufacture of the kitsfollows preferably standard procedures which are known to the personskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

FIG. 1. Parameters determining amplification success. a) Twenty HT29colon carcinoma cells ((ATCC: HTB-38) lanes 1–20) were individuallyisolated and mRNA reverse transcribed in the presence of differentconcentrations of random hexamer primers (lanes 1–5, 80 μM; lanes 6–10,8 μM; lanes 11–15, 0.8 μM; lanes 16–20, 0.08 μM). 1/10 of the cDNA wassubsequently tested for the detection of the ki-ras transcript bygene-specific PCR. b) Influence of the homopolymer tail on sensitivity.A 350 bp TGF-α fragment was isolated, diluted and either dA or dGtailed. Serial dilutions were tested by PCR using poly-dT or poly-dCcontaining primers, respectively, and a primer within the TGF-αsequence. The informative dilutions are shown in duplicates. (lanes 1+2,negative control; lanes 3+4, 10⁻³ dilution; lanes 5+6, 10⁻⁵ dilution).c) FL4-N6 primed and revers transcribed mRNA was dG-tailed and amplifiedusing the CP3+FL4 primers (lanes 1–3) or CP2+FL4 primers at differentannealing temperatures (lane 1+4, 68° C., lane 2+5 65° C., lane 3+6,negative control). d) An identical amount of mRNA as in c) was reversetranscribed using the CFL5cN6 primer, and amplified with the CP2 primer.An equal amount of cDNA as in c) (lane 3+4) resulted in amplification ofa wide range of cDNA fragments as did a 1:200 dilution (lane 1+2) atdifferent annealing temperatures (lane 1+3, 68° C.; lane 2+4, 65° C.;lane 5, negative control).

FIG. 2. Gene specific PCR for β-actin and various MAGE transcripts usingunamplified pooled cDNA of A431 cells as positive control (+) andamplificates of single A431 cells (lane 2–4 and 6–8) that were dividedinto two halves (a+b) before global PCR. Two independent experimentswere performed (lane 1–4 and 5–8) with lane 1 and lane 5 being thenegative controls for the global PCR.

FIG. 3. CGH profiles of two normal leukocytes (red) and two MCF-7 breastcancer cells (blue) of which the genomic DNA was isolated from thesupernatant after mRNA isolation. The chromosomal ratios of the normalcells are within the dashed lines, giving the threshold forsignificance, whereas the profiles of the cancer cells are similar withregard to their chromosomal deletions and amplifications.

FIG. 4. CGH profile of cell B derived from a breast cancer patient withvery small primary tumor (stage T1a). Chromosomal deletions are markedwith a red bar left of and chromosomal gains with a green bar right ofthe chromosome symbol.

FIG. 5. Diagram illustrating the common and differentially expressedgenes of cell B, C and L.

FIG. 6. Hybridisation of cell L (left) and the matrix of positions andnames of immobilized cDNAs. Genes were spotted in duplicates in diagonaldirection, with the blue gene symbols oriented from upper left to lowerright and the red gene symbols oriented from upper right to lower left.

FIG. 7: Immunohistochemical stains of neointima from human coronaryartery in-stent restenosis for v. Giesson (left panel) and smooth musclealpha-actin (right panel). The shown experiment is a representative of 3independent specimen. Bars indicate 100 μm.

FIG. 8: PCR with gene-specific primer for β-actin (lanes 1), EF-α (lanes2) and α-actin (lanes 3) as a control for successful PCR amplificationof the first strand cDNA generated from microscopic tissue specimen.Shown is one representative from each study group (right panel: patientB; left panel: control donor b). The position of three size markers (M)is shown.

FIG. 9: cDNA array analysis. The same array is shown with threeindependent hybridization experiments comparing mRNA isolated fromneointima (panel A) or from control vessel (panel B), and in the absenceof a biological sample (panel C). The cDNA array contained 588 genesincluding nine housekeeping genes and three negative controls [M13 mp 18(+) strand DNA; lambda DNA; pUC18 DNA]. The experiment shown here is arepresentative of hybridization experiments with 10 neointima and 10control specimen. Circles indicate four hybridization signals (A-D)differentially expressed between restenosis and control.

FIG. 10: Transcription profiles of microscopic samples from humanin-stent neointima and control vessels. Each column represents a geneexpression analysis of a single specimen for 53 selected genes. An arrowindicated genes that show significant up- or downregulation in neointimaversus control. Eight highly expressed housekeeping genes are shown onthe bottom. One grey value corresponds to a signal intensity as shown atthe bottom of the figure.

FIG. 11: Verification of differentially expressed mRNAs from cDNA arraysby gene-specific PCR. The size of the expected PCR fragment is indicatedon the right.

FIG. 12: Immunhistochemical staining of neointima from carotid arteryrestenosis for the FKBP12 protein. The experiment shown is arepresentative of three independent experiments. The bars represents adistance of 100 μm. Panel A shows a hematoxylin eosin staining, panelB–D shows staining for FKBP12 of the border zone between healthy mediaand neointima (panel B), of healthy control media (panel C) andneointimal tissue (panel D).

FIG. 13: cDNA array analysis of gene expression. Four Clontech Atlasmicroarrays, containing a total of 2435 human cDNAs, were hybridizedwith cDNA labeled with Dig-dUPT prepared from RNA from in-stentneointima (n=10) and from control media/intima (n=11) as described inMaterials and Methods. Spots indicate the mean of the relativeexpression of the two examined groups. Panel A shows the expression ofall examined genes in this study. Panel B shows expression of the 224differentially expressed genes, that were more than 2.5-fold induced orreduced in neointima and showed a statistical significance p<0.03 in theWilcoxon test. For this presentation, zero value were replaced by avalue of 0.0001, as a zero value is not representable in a logarithmicscale.

FIG. 14: Cluster image showing the different classes of gene expressionprofiles of the two hundred twenty four genes whose mRNA levels weredifferent between neointima and control. This subset of genes wasclustered into four groups on the basis of their expression in differentcell types. The expression pattern of each gene in this set is displayedhere as a horizontal strip. Each column represents the average mRNAexpression level of the examined group. For each gene, the average ofthe mRNA level of neointima (n=10), of control (n=11), of proliferatingCASMCs (n=2) and of blood samples (n=10) normalized to the mRNAexpression level of the housekeeping genes is represented by a color,according to the color scale at the bottom. Group I contained genes onlyexpressed in neointima specimen (FIG. 14A). Group II contained genesexpressed simultaneously in neointima and proliferating CASMCs (FIG.14B). Group III consisted of genes, whose mRNA were expressed inneointima as well as in blood (FIG. 14C). Group IV contained genes,whose mRNA was overexpressed in control specimen (FIG. 14D).

FIG. 15: Expanded view of the transcription factorcluster containing 14genes that were upregulated in neointima versus control and threetranscription factors that were downregulated in neointima. In thiscase, each column represents a single specimen, and each row representsa single gene

FIG. 16: Expanded view of the IFN-γ-associated cluster containing 32genes that were upregulated in neointima versus control. In this case,each column represents a single specimen, and each row represents asingle gene.

FIG. 17 Immunohistochemical stains of neointima from a carotidrestenosis and healthy control media for the IRF-1 protein (left panel:control media; right panel: neointima). The experiment shown is arepresentative of 6 independent experiments.

FIG. 18: Immunohistochemical stain of neointima from a coronary in-stentfor the IRF-1 protein. Panel A shows a hematoxylin eosin staining of theneointimal specimen from in-stent restenosis, panel B shows a stainingfor the smooth muscle cell marker α-actin, panel C shows theimmunohistochemical stain for the transcription factor IRF-1 inneointima from in-stent restenosis and panel D shows immunohistochemicalstain for CD3. The experiment shown here is a representative of threeindependent experiments.

FIG. 19: View of the IFN-γ-associated cluster containing the 32 genesthat were upregulated in neointima versus control compared to expressionin cultured CASMCs and to cultured CASMCs stimulated for 16 h with 1000U/mL IFN-γ. In this case, each column represents a single specimen, andeach row represents a single gene. One grey value corresponds to asignal intensity as shown at the bottom of the figure.

FIG. 20: Double staining of disseminated tumor cells in bone marrow.Cells in small aggregates (of seven and of two cells) in the upper paneland one single cell detected in bone marrow of two different patientswere stained for cytokeratin (red fluorescence) and Emmprin (blue).

FIG. 21: Differential expression of the transferrin receptor (CD71) ontumor cells. DAPI staining of cellular nuclei (left panel), upregulatedCD71 expression is found in tumor tissue (right panel).

FIG. 22: Effect of IFNγ on survival of cultured SMCs. Flow cytometryanalysis of spontaneous (panel A and C) and H₂O₂-induced apoptosis(panel B and D). Cells were double-stained by FITC-labelled Annexin Vand Pi at 6 h after treatment with 100 μmol/l H₂O₂. A representativeanalysis of 5 independent experiments is shown.

FIG. 23. The effect of an IFNγ receptor null mutation on the developmentof neointima in a mouse model of restenosis. (A–D) Representativemicrophotographs of cross-sectioned mouse carotid arteries from wildtype(wt) and IFN-γR^(−/−) knockout (ko) mice are shown for the untreatedartery (control) and the contralateral ligated artery (ligated) at 4weeks after ligation. The van-Giesson staining procedure was used. Thebars represent a lenght of 100 μm. (E) Data from 16 wildtype and 11FN-γR^(−/−) mice are shown as mean±SEM (bars) and analyzed by the t-testfor unpaired samples. The scale gives the thickness of media andneointima in μm. Open columns: control animals before and after carotisligation; filled columns: knockout animals before and after carotisligation. The shaded area indicates the thickness of neointima.

FIG. 24: Flow chart of SSH analysis performed with single cells or smallcell samples.

FIG. 25: Screening of colonies by southern blot using labeled driver andtester as probes. Lane 1–9 colonies obtained after subtraction. Colony#4 was identified as ESE1, an epithelium-specific transcription factor.M=molecular weight marker.

FIG. 26: Differential expression of ESE1 in tumor cells analyzed by PCRand gelectrophoresis. Lane 1–4 single breast cancer cells, 5–7 bonemarrow of healthy donors. M=molecular weight marker.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of scope of the present invention.

EXAMPLE I Generation and Global Amplification of Single Cell cDNA

The amount of mRNA from single cells is too low for direct use inarray-based transcriptome analysis. Total RNA from 50,000 cells (10 μg)was reported to be the detection limit for direct-labelling approaches(Mahadevappa, Nat. Biotechnol., 17, 1134–1136 (1999)). Using a linearamplification step, this number could be reduced to 1000 cells (Luo,Nat. Med., 5, 117–122 (1999)), which is still far beyond applicabilityfor the study of micrometastatic cells. Thus reverse transcription ofmRNA and amplification of the cDNA is necessary. Key is the developmentof an unbiased global amplification procedure. In a simplified manner,this approach consists of four basic steps: (1) isolation of the mRNA onoligo-dT-coated solid support, (2) cDNA synthesis using random primerscontaining a 5′-oligo-dC (or dG) flanking region, (3) 3′tailing reactionwith dGTP (or dCTP) generating a 3′-oligo-dG flanking region, followedby (4) single primer-based amplification using a primer hybridizing tooligo-dG (or -dC) flanking regions of the cDNA molecules. In order tofulfil these four basic steps and to obtain high sensitivity andreliability for cDNA synthesis, 3′-tailing and pCR amplification, tRNAand rRNA had to be removed.

Furthermore, concentrations of random primers were 2000–8000-timeshigher for cDNA synthesis compared to previously desribed oligo-dT-basedapproaches (Brady, Methods. Enzymol., 225, 611–623 (1993); Trumper,Blood, 81, 3097–3115 (1993)), who employed 10 nM cDNA synthesis primers.Twenty HT29 colon carcinoma cells (ATCC: HTB-38) were individuallyisolated and processed. After cell lysis in cDNA synthesis buffercontaining the detergent Igepal, groups of five cells were formed andreverse transcribed with four different concentrations of random cDNAsynthesis primers. By gene-specific RT-PCR cDNA synthesis was tested foreach concentration. FIG. 1 a shows that higher concentrations of randomprimers for cDNA synthesis lead to increased detection rates of specifictranscripts (e.g. ki-ras). Surplus primer, being an effective competitorof the subsequent tailing and amplification reaction, was, therefore,preferably removed prior to both steps. Equally, high dNTPconcentrations improved cDNA synthesis but interfered with thesubsequent tailing reaction and needed to be removed. Standardcacodylate-containing tailing buffer interfered with the following PCRand was replaced with a KH2PO4 buffer of low ionic strength (Nelson,Methods Enzymol., 68, 41–50 (1979). Capturing of mRNA on oligo-dT coatedmagnetic beads provided for simple handling during mRNA isolation andbuffer exchange steps. In the following, the isolation of single cells,mRNA isolation, cDNA synthesis and 3′-tailing is briefly described andexemplified.

Tumor cells were isolated from bone marrow as described (Klein, Proc.Natl. Acad. Sci. USA, 96, 4494–4499 (1999)). Briefly, viable bone marrowsamples were stained for 10 min. with 10 μg/ml monoclonal antibody3B10-C9 in the presence of 5% AB-serum to prevent unspecific binding.3B10-positive cells were detected with B-phycoerythrin-conjugated goatantibody to mouse IgG (The Jackson Laboratory) and transferred toPCR-tubes on ice. Oligo-dT beads were added, the cells lysed in 10 μllysis buffer (Dynal), tubes rotated for 30 min. to capture mRNA. 10 μlcDNA wash buffer-1 (Dynal) containing 0.5% Igepal (Sigma) was added andmRNA bound to the beads washed in cDNA wash buffer-2 (50 mM Tris-HCl, pH8,3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, supplemented with 0.5% Tween-20(Sigma)), transferred to a fresh tube and washed again in cDNA washbuffer-1 to remove any traces of LiDS and genomic DNA. mRNA was reversetranscribed with Superscript II Reverse Transcriptase (Gibco BRL) usingthe buffers supplied by the manufacturer supplemented with 500 μM dNTP,0.25% Igepal, 30 μM Cfl5c8 primer (5′-(CCC)₅ GTC TAG ANN(N)₆-3′) and 15μM CFL5cT (5′-(CCC)₅ GTC TAG ATT (TTT)₄ TVN, at 44° C. for 45 min.Samples were rotated during the reaction to avoid sedimentation of thebeads. cDNA remained linked to the paramagnetic beads via the mRNA andwashed once in the tailing wash buffer (50 mM KH₂PO₄, pH 7.0, 1 mM DTT,0.25% Igepal). Beads were resuspended in tailing buffer (10 mM KH₂PO₄,pH 7.0, 4 mM MgCl₂, 0.1. mM DTT, 200 μM GTP) and cDNA-mRNA hybrids weredenatured at 94° C. for 4 min, chilled on ice, 10 U TdT (MBI-Fermentas)added and incubated at 37° C. for 60 min or 37° C., 60 min and 22° C.over night. After inactivation of the tailing enzyme (70° C., 5 min),PCR-Mix I was added consisting of 4 μl of buffer 1 (Roche, Taq longtemplate), 3% deionized formamide (Sigma) in a volume of 35 μl. Theprobes were heated at 78° C. in the PCR cycler (Perkin Elmer 2400), PCRMix II, containing dNTPs at a final concentration of 350 μM, CP2 primer(5′-TCA-GAA-TTC-ATG-CC-CCC-CCC-CCC-CCC-3′, final concentration 1.2 μM)and 5 Units of the DNA Poly-Mix was added, (Roche, Taq Long Template) ina volume of 5 μl for a hot start procedure. Forty cycles were run at 94°C., 15 sec, 65° C., 30° C., 68° C., 2 min for the first 20 cycles and a10 sec-elongation of the extension time each cycle for the remaining 20cycles, and a final extension step at 68° C., 7 min. These PCRamplification conditions differ substantially from Brail, Mut. Res.Genomics, 406, 45–54 (1999). Annealing temperature in Brail is only 42°C. for 2 min in contrast to the 65° C. applied in this example of methodof invention.

Tailing efficiency as well as the sensitivity of the subsequent PCR ofpoly-dA- and poly-dG-tailed sequences was assessed using a defined cDNAfragment with a homopolymer tail of either poly-dA or poly-dT. Thepoly-(dA) and poly-(dG)-tailed fragments were diluted and then amplifiedby PCR using equal amounts of poly(dT) and poly(dC) primers,respectively. In these experiments poly-C primers binding to poly-Gtails were found to be at least 100-times more sensitivity than poly-Tprimers on poly-dA tails (FIG. 1 b compare lanes 1,2 to 3,4)

Various cDNA synthesis primers sharing the same poly-dC flanking regionin combination with random hexamers (N6), octamers (N8), oligo-dT (dT)₁₅alone or in combination were compared. All worked well and reliably. Thebest results were obtained with a combination of poly-dC-N8 andpoly-dC-(dT)₁₅ primers (data not shown).

The most dramatic improvement was obtained when only one primer (FIG. 1c) was used for global PCR instead of two (FIG. 1 d). The cDNA synthesisprimer consisted of a 3′ random hexamer and flanking region either apoly-dC stretch (CFl5c) or a flanking sequence of all four bases(Fl4N6). Two poly-dC binding primers were tested in combination with anadditional primer binding to Fl4 complementary sequence (FIG. 1 c). Useof an additional primer (FL4) to the poly-dC binding primers (CP2, CP3)prevented amplification (FIG. 1 c, lanes 1,2 and 4,5). This is likelydue to the high primer concentrations required for optimal sensitivity.The use of the CP2 primer alone resulted in amplification of a widerange of cDNA molecules (0.2–3 kB). Even highly diluted cDNA (1:200) wasstill sufficient for global amplification (FIG. 1 d).

EXAMPLE II Transcriptome Analysis of Singi Cells: Specificity,Reproducibility, Sensitivity, and Suitability for cDNA Array Analysis

Isolated single cells from cultured cell lines were analyzed by theoptimized protocol for cDNA synthesis, tailing and amplification. Atotal of 100 single cells have so far been successfully tested forβ-actin and EF-1α expression by gene-specific PCR (data not shown).cDNAs for housekeeping genes were found in a sufficient copy number percell to be relatively independent of the region used for specificamplification in the secondary PCR. For less abundant transcripts, itwas noted that the size of the chosen coding sequence determineddetection rates. Highest sensitivity was obtained with the two primersbeing separated by less than 200 bp (data not shown).

The PCR amplificates from single cells were tested for suitability ofcDNA array analysis. For this purpose, the obtained cDNA wasDig(Digoxigenin)-labeled. Dig-UTP was incorporated by PCR. Forexpression profiling 0.1–1 μl of the original PCR amplified cDNAfragments were used for reamplification in the presence ofdigoxigenin-labeled dUTP (Boehringer Mannheim), 50 μM dig-dUTP, 300 μMdTTP, and other dNTPs at a final concentration of 350 μM.Reamplification conditions were essentially as described above,modifications were the use of 2.5 Units of the DNA Poly Mix. Initialdenaturation at 94° C. for 2 min. followed by 12 cycles at 94° C., 15sec, 68° C., 3 min and a final extension time of 7 min. Specifictranscripts were detected using 1 μl of a 1/10 dilution of the originalPCR to a final volume of 10 μl.

The specificity of the hybridization of digoxigenin-labeled probes isdepicted in Table 1, where the expression pattern of genes from singlecells of different histogenetic origin are shown. Cells were MCF-7 (ATCCNumber HTB-22), A431 (ATCC Number CRL-1555), K-562 (ATCC NumberCCL-243), JY (International Histocompatibility Workshop: IHW9287). Onlythe MCF-7 and A431 cell expressed the cytokeratin genes, markers fortheir epithelial origin, whereas the erythroleukemia K562 cell andEBV-transformed B cell JY expressed genes of a haematopoetic origin,including CD33, CD37, CD38, and kappa light chain in the B cell. Inaddition, the testis- and tumor-specific MAGE genes were highlyexpressed in all cancer cells but not the virally transformed B cell.These data show that single cell PCR amplificates are useful for cDNAarray analysis and produce cell type-specific gene expression patternsof single cells.

TABLE 1 Expression of histogenetically informative genes by single cellsderived from different tissues. MCF-7 A431 K562 JY Aktin + + + +EF-1a + + + + CK7 + + − − CK10 − + − − CK13 − + − − CK18 + + − −CK19 + + − − EGP + + − − CD33 − − + + CD37 − − + + CD38 − − + − Kappa −− − + Vimentin − + + − α-6 Integrin + − − − β-1 Integrin + − − − β-2Integrin − − − + β-4 Integrin − − + − β-7 Integrin − − − + FAK + − − −Mage1 + − + − Mage2 + + + − Mage3 + − + − Mage6 + − + − Mage12 + + + −

Individual cells grown in culture were isolated, cDNA synthesized,amplified and hybridized to an array of histogenetically informativegenes. Cells were from the following cell lines MCF-7 (breast cancer);A431 (epidermoid carcinoma); K562 (chronic myeoloid leukemia); JY(Epstein-Barr virus transformed B cell line).

In order to assess reproducibility, the expression pattern of four MCF-7cells were compared using a cDNA array Generation 4 with 110 differentgenes (Table 2). Custom made cDNA arrays were prepared as follows. cDNAswere PCR-amplified with gene-specific primers from human cDNA, PCRamplificates were gel-purified and 15 ng DNA per amplificate was spottedonto nylon membranes (Boehringer) using a BioGrid spotting roboticdevice (Biorobotics). DNA Macroarrays were termed Generation 4 andGeneration 5 (see herein below).

Filter Generation 4: Spotted Genes were:

Protein Name HUGO Name Protein Name HUGO Name Cytokeratin 7 KRT7 slapSLA Cytokeratin 8 KRT8 p21 CDKN1A Cytokeratin 10 KRT10 p68 Cytokeratin13 KRT13 p27 CDKN1B Cytokeratin 18 KRT18 Eck EPHA2 Cytokeratin 19 KRT19P33 ING1 Cytokeratin 20 KRT20 B61 EFNA1 Emmprin II BSG p53 III TP53MT1-MMP MT1-MMP E-Cad CDH1 MT2-MMP MT2-MMP p53 IV TP53 MT3-MMP MT3-MMPP-Cad CDH3 MT4-MMP MT4-MMP p57 CDKN1C TIMP1 TIMP1 N-Cad CDH2 TIMP2 TIMP2Cyclin D CCND1 TIMP4 TIMP4 c-myc I MYC MMP1 MMP1 Gas1 GAS1 uPA PLAUc-myc II MYC uPA-Rezeptor PLAUR Ki-67 MKI67 PAI1 PAI1 RB RB1 PAI2 PAI2b-Aktin ACTB CathepsinB CTSB HTK TK1 CathepsinD CTSD EF-1a EEF1A1CathepsinL CTSL RAD 51 RAD51 Stromelysin1 MMP3 A20 TNFAIP3 Stromelysin3MMP11 Nck NCK1 Gelatinase A MMP2 BCL-2 BCL2 Gelatinase B MMP9 pBSMatrilysin MMP7 GAPDH GHPDH Cystatin 1 CSTA hEST TERT Cystatin 2 CSTBMage 1 MAGEA1 Cystatin 3 CST3 TSP-1 THBS1 ADAM 8 ADAM8 Mage 3 MAGEA3ADAM 9 ADAM9 mrp-1 ABCC1 ADAM 10 ADAM10 Mage 4 MAGEA4 ADAM 11 ADAM11mdr-1 ABCB1 ADAM 15 ADAM15 Mage 6 MAGEA6 ADAM 20 ADAM20 DEP-1 PTPRJ ADAM21 ADAM21 Mage 12 MAGEA12 TACE ADAM17 PTP-μ PTPRM a4-Integrin ITGA4Mage1F MAGEA1 a5-Integrin ITGA5 Creatin Kinase CKM a6-Integrin ITGA6Mage2F MAGEA2 av-Integrin ITGAV Mage 4F MAGEA4 GFP Mage3F MAGEA3beta-Actin ACTB Mage 12F MAGEA12 b1-Integrin ITGB1 CD16 FCGR3Ab2-Integrin ITGB2 TGF-a TGFA b3-Integrin ITGB3 CD33 CD33 b4-IntegrinITGB4 TGF-b TGFB1 b5-Integrin ITGB5 CD34 CD34 b7-Integrin ITGB7 VEGFVEGF p15 CDKN2B CD37 CD37 Fak PTK2 IGF-I IGF1 p16 CDKN2A CD38 CD38Ramp-1 kappa IGKC CD40 CD40 TGF-b R.II TGFBR1 Ramp-2 lambda IGLC1 CD45II PTPRL IGF-RI IGFR1 EMM I BSG Vimentin VIM CD83 CD83 IGF-RII IGFR2 GFPEGP-1 M4S1 pBS MUC 18 MCAM erb B2 ERBB2 DP-I DSP TCR TCRA PHRIP PHLDA1TGF-b Rez.I TGFBR1 CEA CEA EF-1a EEF1A1

TABLE 2 Commonly and differentially expressed genes of four single MCF-7cells. 4/4 3/4 2/4 1/4 EF-1a CK19 Beta-4-Integrin CK10 GAPDH TIMP-1Beta-5-Integrin CK13 b-Actin Cathepsin B P53 ADAM 9 CK7 Cathepsin DCreatin Kinase ADAM 15 CK8 Cathepsin L ADAM17 (TACE) CK18 ADAM 10 p16CK20 c-myc p21 Alpha 6-Integrin p27 Beta1-Integrin p33 Fak ki-67 EMMPRINhTK u-PAR E-cadherin Matrilysin IGF-R I Cyclin D1 IGF-R II Eck TGF-betaEpCAM VEGF Mrp-1 DP-I PHRIP

Heterogeneity of gene expression of individual cells derived from thesame cell clone. Four MCF-7 cells isolated from cell culture wereanalyzed by single cell analysis of gene expression. Listed are thetranscripts that were detected in all four single cells (4/4), three offour (3/4), two of four (2/4), and one of four (1/4). 18/46 (39%)expressed genes were detected in all cells. 61% genes could only foundin a portion of the four cells. 63 genes were negative for all cellstested.

46 genes (42%) were expressed in at least one cell and 63 (58%) werenegative for all four cells. Eighteen of the 46 (39%) expressed geneswere detected in all four cells whereas the remaining 29 (61%) werefound to be heterogeneously expressed. To evaluate whether thisheterogeneity was due to intercellular variation or is an artifact ofthe technique, it was tested whether disparity is also observed with thecDNA of a single A431 cell that was split for two separate PCRamplifications. In a first experiment, gene-specific PCRs with theglobally amplified PCR products obtained from 50% of single cell cDNA(FIG. 2) were performed. For comparison, cDNA isolated from a pool of500.000 A431 cells were diluted to such an extent that the intensity ofthe β-actin band was similar to that obtained with 50% of the singlecell cDNA. After 32 cycles and with a cDNA amount corresponding to about10.000 cells, the β-actin signal of the pool control and 50% of thesingle cell cDNA reached the plateau phase of amplification. As shown inFIG. 2, the variation between two cDNA halves of the same cell was verylow. In two independent experiments, each half (a+b) from six A431 cellsyielded β-actin bands of similar intensity.

In order to test the reliability of the global amplification of thecDNA, a second gene sequence-specific PCR amplification was perfomed. Asthe efficiency of gene-specific PCR amplification is known to be primersequence-dependent, the amplification of MAGE transcripts was tested,which are very demanding with respect to primer design (Kufer,WO98/46788 (1998); Serrano, Int. J. Cancer 83, 664–669 (1999)). Thelevel of MAGE expression determined by sequence specific PCR wasconsistently lower than that of beta-actin. The relative abundance ofMAGE transcripts in split single cell samples after global PCRamplification of the cDNA (FIG. 2, lanes 2–4 and 6–8) was comparable tothat of the control sample from unamplified cDNA from pooled cells (FIG.2, +). In 4 out of 6 cases, the results were identical for both halvesof the cDNA. The lack of any MAGE transcript in cell half 7a and 8b mostlikely indicates an unequal distribution of the cDNAs between the twohalves.

The observed sequence-independent amplification is characteristic of thepoly-dC primer, which contains fifteen cytosine residues and thereforeintroduces primer binding sites with equally high CG-content. Theexperimental conditions suited for such a primer, i.e. high annealingtemperature (65° C.) in the presence of 3% denaturing formamide, lead,to a remarkable reproducibility and did not introduce major quantitativechanges to the single cell transcriptome.

Amplificates from split single cell cDNAs and, as control, cDNA from5,000 pooled cells were labeled and hybridized to a cDNA arrayrepresenting 193 different genes. Most transcripts could be detectedwith both halves of the single cell amplificates (Tab. 3).

Table 3: Gene expression patterns of single cells split in two pols ofcDNA prior to global PCR compared to pooled cDNA of 5000 cells.

TABLE 3

The cDNAs of two single cells were split prior to PCR amplification andcompared to a cDNA pool derived from 5000 cells. All cDNAs wereamplified by global PCR and analyzed by hybridization to a cDNA array.The gene expression profiles of the corresponding halves (1.1 and 1.2;2.1 and 2.2) are juxtaposed to the cell pool (+). The genes are listedaccording signal strength (the darker, the stronger) and detection inboth halves of the same cell. The filter used was Generation 5, genesand protein names are listed below (for preparation of said Generation 5filter, see herein above (Generation 4 filter)).

Generation 5 Filter:

Protein HUGO A20 TNFAIP3 a4-Int ITGA4 a5-Int ITGA5 a6-Int ITGA6 ADAM10ADAM10 ADAM15 ADAM15 ADAM21 ADAM21 ADAM9 ADAM9 Auto-Ag SHGC-74292 av-IntITGAV Axl AXL b1-Int ITGB1 b2-Int ITGB2 b3-Int ITGB3 b4-Int ITGB4 b5-IntITGB5 B61 EFNA1 b7-Int ITG7 BA46 MFGE8 BAG1 BAG1 b-Aktin ACTB b-CaseinCSN2 Bcl-2 BCL2 Bcl-xl BCL2L1 b-micro MSMB BTG-3/ANA BTG3 CalmodulinCALM1 Cathepsin B CTSB Cathepsin D CTSD Cathepsin L CTSL CD16 FCGR3ACD24 CD24 CD33 CD33 CD34 CD34 CD37 CD37 CD38 CD38 CD40 TNFRSF5 CD44 CD44CD45 PTPRC CD83 CD83 CEA CEA CK10 KRT10 CK13 KRT13 CK18 KRT18 CK19 KRT19CK7 KRT7 CK8 KRT8 Claud1 CLDN1 Claud3 CLDN3 Claud7 CLDN7 c-myc MYCBPCyclin D1 CCND1 Cystatin A CSTA Cystatin B CSTB Decoy-R2 TNFRSF10DDecoy-R3 TNFRSF6B DEP-1 PTPRJ DP-1 DSP E2F6 E2F6 E-Cad CDH1 Eck EPHA2EF1a EEF1A1 EGP1 M4S1 Emmprin BSG EPC-1 PEDF erbB2 ERBB2 Ese1b/ELF3 ELF3Fak PTK2 FGFR1 FGFR1 FGFRII FGFR2 Gadd45 GADD45A GAPDH GHPDH Gas1 GAS1Gas6 GAS6 GFP hEST TERT Hevin HEVIN HTK TK1 ICAM ICAM IGF RI IGFR1 IGFRII IGFR2 Kappa IGKC Ki67 MKI67 KIA169 Lambda IGLC1 lot1/hZAC Hs.75825Mage1 MAGEA1 mage12 MAGEA12 Mage2f MAGEA2 Mage4 MAGEA4 MAT8 PLML Mdr-1ABCB1 MLN62 TRAF4 MLN64 TRAF4 mrp-1 ABCC1 MT1-MMP MT1-MMP Muc 18 MCAMN-Cad CDH2 Nck NCK1 p15 CDKN2B p16 CDKN2A p21 CDKN1A p27 CDKN1B p33 ING1p53III TP53 (III) p53IV TP53 (IV) p57 CDKN1C p68 PAI-2 PAI2 pBS P-CadCDH3 Phospholipase PLD1 Phrip PHLDA1 PIP PIP Prohibitin PHBProst.Spec.Home Hs.73189 o. Prost.Spec.Trans TGM4 glu Prost.Spec.Uro.UPK3 Prothym alpha PTMA PSA KLK3 PTHrP PTP-μ PTPRM RB RB1 rfx-1 RFX1Slap SLA Stromelysin 1 MMP3 Survivin API4 TACE ADAM17 TCR TCRA TGF-alphaTGFA TGF-beta TGFB1 TGFB-RI TGFBR1 TGFB-RII TGFBR2 TIE-2/Tek TEK TIG3RARRES3 Timp1 TIMP1 TMP21 TMP21 TSP-1 THBS1 Tubulin-a TUBA Ubiquitin UBuPA PLAU uPA-R PLAUR VEGF VEGF Vimentin VIM VLDLR VLDLR ZNF217 ZNF217Hs.46452

A total of 148 signals were obtained for the four cDNA halves. Of these,95 (64%) were found in the corresponding halves, whereas 53 (36%) werefound in only one half. Out of the 53 single positive signals 46 (87%)represented very low-abundant transcripts, with 26 (49%) not detectableand 20 (37%) only weakly expressed in the control of pooled cells. Sevengenes (AXL, BAG1, BCL2L1, SHGC-74292, B61, TGFBR2 and ABCC1) wereexclusively detected in the pooled sample, though with a rather weaksignal. In contrast, 33 genes were only found in the half-cellexperiments but not in the control. The signal intensity of the bothhalves was quite similar, with 55% and 76% of the signals having thesame strength in the corresponding halves. Signals that were notidentical in two corresponding halves may arise from of a non-randomdistribution of cDNA fragments prior to PCR. Particularly transcriptspresent in low (<10) copy number may be subject to such a distributioneffect which, however, may not be obtained if samples are not split.

EXAMPLE III Combined Transcriptome and Genome Analysis from Single Cells

A method of CGH (comparative genomic hybridization) analysis of singlecells (SCOMP) was recently described (Klein, Proc. Natl. Acad. Sci. USA,96, 4494–4499 (1999)). Using this method, a tumor cell can unambiguouslybe identified by its chromosomal aberrations. It was therefore attemptedto isolate both genomic DNA and mRNA from the same cell. Isolated singlecells were lysed in 10 μl lysis buffer (Dynal) and tubes rotated for 30min. to capture mRNA. 10 μl cDNA wash buffer-1 (50 mM Tris-HCl, pH 8,3,75 mM KCl, 3 mM MgCl₂, 10 mM DTT, supplemented with 0.5% containing 0.5%Igepal (Sigma)) was added and mRNA bound to the beads washed in cDNAwash buffer-2 (50 mM Tris-HCl, pH 8,3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT,supplemented with 0.5% Tween-20 (Sigma)), transferred to a fresh tubeand washed again in cDNA wash buffer-1 to remove any traces of LiDS andgenomic DNA. mRNA was reverse transcribed with Superscript II ReverseTranscriptase (Gibco BRL) using the buffers supplied by the manufacturersupplemented with 500 μM dNTP, 0.25% Igepal, 30 μM Cfl5c8 primer (SEQ IDNO:9) (5′-(CCC)₅ GTC TAG A (N)₈-3′) and 15 μM CFL5cT (SEQ ID NO:10)(5′-(CCC)5 GTC TAG ATT (TTT)₄ TVN, at 44° C. for 45 mm. Samples wererotated during the reaction to avoid sedimentation of the beads. Primersused and mentioned in FIGS. 1 c and d were Cfl5cN6 (SEQ ID NO:8)(5′-(CCC)₅ GTC TAG A (N)₆-3′) and FL4N6 (SEQ ID NO:6) 5′-TTT CTC CTT AATGTC ACA GAT CTC GAG GAT TTC (N)₆-3′). cDNA remained linked to theparamagnetic beads via the mRNA and washed once in the tailing washbuffer (50 mM KH₂PO₄, pH 7.0, 1 mM DTT, 0.25% Igepal). Beads wereresuspended in tailing buffer (10 mM KH₂PO₄, pH 7.0, 4 mM MgCl₂, 0.1 mMDTT, 200 μM GTP) and cDNA-mRNA hybrids were denatured at 94° C. for 4mm, chilled on ice, 10 U TdT (MBI-Fermentas) added and incubated at 37°C. for 60 mm or 37° C., 60 mm and 22° C. over night. After inactivationof the tailing enzyme (70° C., 5 mm), PCR-Mix I was added consisting of4 μl of buffer 1 (Roche, Taq long template), 3% deionized formamide(Sigma) in a volume of 35 μl. The probes were heated at 78° C. in thePCR cycler (Perkin Elmer 2400), PCR Mix II, containing dNTPs at a finalconcentration of 350 μM, CP2 primer (SEQ ID NO:14)(5′-TCA-GAA-TTC-ATG-CCC-CCC-CCC-CCC-CCC-3′, final concentration 1.2 μM)and 5 Units of the DNA Poly-Mix was added, (Roche, Taq Long Template) ina volume of 5 μl for a hot start procedure. Forty cycles were run at 94°C., 15 sec, 65° C., 30° C., 68° C., 2 mm for the first 20 cycles and a10 sec-elongation of the extension time each cycle for the remaining 20cycles, and a final extension step at 68° C., 7 mm. PCR primers used inFIG. 1 c were CP3 (SEQ ID NO:15) (5′-GCT GAA GTG GCG AAT TCC GAT GCC(C)₁₂-3′) and FL4 (SEQ ID NO:16) (5′-CTC CTT AAT GTC ACA GAT CTC GAG GATTTC-3′).

The supernatants from the cell lysis and all washing steps (cDNA washbuffer 1 and 2) of the mRNA isolation were collected (total volume 60μl). After transfer to a silanised tube the genomic DNA was ethanolprecipitated overnight at −20° C. in the presence of 20 μg glycogen(Roche). All subsequent steps were performed as published (Klein,(1999), loc. cit.).

A major concern was incomplete precipitation of genomic DNA eventuallyleading to losses of DNA as seen with chromosome deletions in cancerouscells. However, experiments with cells of a defined karyotype clearlyshowed that either the cellular DNA was totally lost (30% of cases) orcompletely precipitated (70%) (data not shown). The complete recovery ofgenomic DNA may be due to the fact that interphase chromosomes areextensively interwoven so that either all or none is precipitated. Theloss of all DNA is probably introduced by the change of reaction tubesduring the separation of genomic DNA and mRNA. The karyotypes of twonormal and two MCF-7 breast cancer cells whose DNA had been precipitatedare shown in FIG. 3. The profiles of the two normal cells showed nosignificant deviation from the midline while the multiple genomicaberrations of the two MCF-F7 cells were almost identical. Hence,malignant EpCAM-positive cells can be unambiguously distinguished bytheir genomic phenotype from normal EpCAM-positive cells in the bonemarrow. This is of particular importance since EpCAM-expression isinsufficient proof for the (malignant) identity of tumor cell(s) in bonemarrow samples. It has to be noted that healthy donors also showed0.5–5% ″3 3B1O-C9-positive cells (3B10-C9, Prof. Judy Johnson, Institutefor Immunology, Munich) is a high affinity mAb against EPCAM) whendetermined by immunofluorescence.

EXAMPLE IV Activity-related Gene Expression in Three MicrometastaticCells

Single tumor cells were isolated from three patients with differenttumors and disease stages. The first patient (C) had a 10-year historyof cervical carcinoma and presented with a suspicious finding on chestx-ray. In the second patient (L), an adenocarcinoma of the lung hadrecently been diagnosed which was post-operatively staged as pT2, N3,M0. The bone marrow sample was obtained during the anesthesia prior tothe operation. The third sample was aspirated from the pelvic crest of a31-year old breast cancer patient (B) whose disease was in the stagepT1a, pN1a (1/18), M0. Because of a local relapse, the histological G3grading, and finding of one cytokeratin-positive cell in the bonemarrow, this patient received high-dose chemotherapy (HD). The bonemarrow sample was taken one month after completion of HD. SCOMP wasperformed with all three cells and showed multiple chromosomalaberrations verifying the cancerous origin of cells (Tab. 4).

TABLE 4 Genomic aberrations of 3B10-C9-positive cells isolated from bonemarrow of a three patients with cervical carcinoma (C), lung cancer (L)and breast cancer (B). Cell 1p 1q 2p 2q 3p 3q 4p 4q 5p C G G L L G L L GB G G Cell 5q 6p 6q 7p 7q 8p 8q 9p 9q C G/L L L L G G G G L G L B L LCell 10p 10q 11p 11q 12p 12q 13q 14q 15q C G L L L L L G L L B L L G G GCell 16p 16q 17p 17q 18p 18q 19p 19q 20p C L G L L L G L L B Cell 20q21p 21q 22p 22q Xp Xq Y C L L L G G G B G G

Summary of the CGH-data obtained from the three micrometastatic cells.Losses (L) and gains (G) on the small (p) and long (q) arm of eachchromosome are given for each cell.

The cell from patient B, who had the least advanced disease, showed thelowest extent of chromosomal changes (FIG. 4).

mRNA was isolated from all three cells and samples generated for SCAGEas described above. As control, the procedure was performed without theaddition of a cell. cDNA amplificates were hybridized to Clontech Cancer1.2 filters and to newly generated arrays (Axxima A6, Martinsreid)comprising a total of 1,300 genes.

Non-radioactiv hybridization to nylon filters was carried out asfollows:

15 ng of the different PCR-amplified and subcloned cDNA fragments werespotted on positively charged nylon filters by Axxima A G, Martinsried.Filters were pre-hybridized overnight in the presence of 50 μg/ml E.coli and 50 μg/ml pBS DNA in 6 ml Dig-easy Hyb buffer (RocheBiochemicals). 9 μg of labeled PCR products from single cells were mixedwith 100 μg herring sperm, 300 μg E. coli genomic DNA and 300 μg,denatured for 5 min at 94° C., added to 6 ml Dig-easy hybridizationbuffer and hybridized for 36 hours. Stringency washes were performedaccording to the Roche digoxigenin hybridization protocol adding twofinal stringency washes in 0.1×SSC+0.1% SDS for 15 min at 68° C.Detection of filter bound probes was performed according to theDigoxigenin detection system protocol supplied with the kit (Roche).

Only three genes had to be excluded from analysis because a signal wasobtained in at least one of the negative controls. These genes were theVHL-binding protein, caspase 10, TGF-β and hemoglobin α. The number ofpositive signals ranged from 5.3% (70/1313), 7.0% (92/1313) to 11.8%(155/1313) for cells from patients B, C, and L respectively. Thesenumbers were considerably lower than those from single in vitro-growncarcinoma cells where signals were obtained with 10–20% of genes (datanot shown). All three tumor cells expressed genes known to play a rolein regulation of proliferation, replication or growth arrest (FIG. 5;Tab. 5).

TABLE 5 Upregulated genes implied in cell cycle status in cells C, L andB. Role in cell cycle C B L Positive RFC3 RFC3 RFC3 regulators LIG1 LIG1STK12 STK12 P2G4 P2G4 RFC2 RFC2 ADPRT ADPRT S100A4 S100A4 CCNA (cyclinA) CDC25 MKI67 (Ki-67) VRK2 CENPF DYRK4 D123 PRIM1 PIN1 PRKDC (DNA-PK)EB1 CHD3 CDC27HS CALM1 UBL1 TOP2A HMGIY HDAC3 RBBP4 Negative CDKN1A(P21) CDKN1A (P21) regulators ING1 ING1 DDIT1 (GADD45) CDKN2A (P16)

Cells C and B expressed several positive regulators of the cell cycle,while only B and L expressed cell cycle inhibitors.

Cell C expressed the highest number of genes important for cell cycleprogression, including cyclin A (CCNA), EB1, RC2, P2G4, PIN1, RBBP4 andCENPF. As most of these genes are tightly transcriptionally regulatedand their mRNAs are rapidly degraded as cell division progresses, theirexpression not only indicates that cell C was engaged in cycling but canbe faithfully captured in this activity by SCAGE.

Cell B expressed a number of genes important for replication as well ascell cycle inhibition. The pattern of transcripts suggests that the cellwas in a state of DNA repair. The coexpression of GADD45 (DDIT1) and p21(CDKN1A) are indicative for growth arrest (Smith, Science, 266,1376–1380 (1994)). Likewise, the expression of positive cell cycleregulators such as DNA-PK, RFC2, LIG1, ADPRT and PRIM1 has beenimplicated in DNA repair (Lindahl, Science, 286, 1897–1905 (1999);Barnes, Cell, 69, 495–503 (1992), Mossi, Eur. J. Biochem., 254, 209–216(1998); Lee, Mol. Cell Biol. 17, 1425–1433 (1997)). As this cellsurvived an alkalyting, genotoxic high dose chemotherapy its expressionprofile may be interpreted as if re-entry into cell cycle was obviated.This interpretation is supported by the expression of pro-apoptoticgenes such as caspase-6 and BAD that were only found with this cell.Execution of apoptosis in this cell may however be counteracted byexpression of survivin (API4) (FIG. 5; Tab. 5).

The transcriptome obtained from cell L showed traits compatible with itsengagement in dissemination and EMT. While gene expression of cell L didnot resemble that of a cycling or DNA-repairing cell (see above) its 84differentially expressed genes are mostly involved in cytoskeletalreorganization, cell adhesion and extracellular proteolytic activity(Tab. 6; FIG. 6).

TABLE 6 Upregulated genes in cell L indicative for an Invasivephenotype. cytoskeletal organization Adhesion proteolytic activityCytokeratin 2 Integrin alpha 3 Cathepsin B Cytokeratin 6 Integrin alphav Cathepsin D Cytokeratin 7 Integrin beta 2 Cathepsin L Cytokeratin 8Integrin beta 3 MMP7 Cytokeratin 10 Integrin beta 7 MT1-MMP Cytokeratin13, 15, 17 MT2-MMP Cytokeratin 18 Cytohesin 1 uPA Cytokeratin 19 Focaladhesion kinase uPA-R Vimentin Desmoglein 2 ADAM 8 Beta-actin E-cadherinADAM 15 CD9 ADAM 17 RhoA Bikunin RhoB Rho-GDI2 Cystatin 2 A-raf EMMPRINRAP-1A Cdc42 Rac1 P160 ROCK Ste20-like kinase Beta-catenin

The present study analyzed for the first time cellular activities ofindividual tumor cells derived from the bone marrow of cancer patients.Cell C was derived from a cervical carcinoma patient who presented withlung metastasis after a ten-year latent period. This cell was found inproliferation. Cell B was from bone marrow of a breast cancer patientwith a rather small primary cancer who had received high dosechemotherapy because of the apparent aggressiveness of her tumor. Thiscell showed relatively few and discrete genomic changes, a finding thatis of particular interest with regard to the genomic changes requiredfor dissemination. Moreover, this cell must have survived four cycles ofa regular chemotherapy consisting of Epirubicin and Taxol in addition toa high-dose chemotherapy regimen involving alkylating agents. Theobtained expression profile is diagnostic for growth arrest and ongoingDNA repair.

Most informative with respect to the process of dissemination was thetranscriptome of cell L. Detected in a bronchial cancer patient withoutclinically manifest metastasis, this cell expressed many genes encodingproteins involved in active migration and invasion. Most of theactivation cascade of the uPA system was found expressed, consisting ofthe cathepsin B, D, L, the uPA receptor and uPA itself. Likewise, genesinvolved in organizing filopodia, lamellipodia and stress fibers, theRho family members RhoA and B, Rac1, Cdc42 and p160 rock, and genesencoding several adhesion molecules were upregulated in this cell. Itscytoskeleton seemed to undergo remodeling as shown by expression of manycytokeratins and vimentin, a marker for EMT.

It is noteworthy that the number of transcripts in single cells isolatedfrom cultured cell lines was considerably lower than that frompatient-derived tumor cells. This difference may speak for a tighter invivo control of transcription that may become more relaxed when cellsare grown in cell culture, e.g., by increased DNA demethylation.Expression analysis of ex-vivo specimen might therefore be much moreinformative than studies on cell lines. The minimal number of cells thathas been used for cDNA array analysis so far was in the range of 1,000cells (Luo (1999), loc. cit.). The sensitivity of the arrayhybridization might be further increased by longer immobilized cDNAfragments (fragment length on Clontech arrays is about 200 bp), and theamount of information obtained by using glass chips with higher densityand complexity. Although the present study analyzed only 1,300 genes,one has to consider that expression of only nine proteins has thus farbeen reported for micrometastatic cells. These proteins are ErbB2,transferrin receptor, MHC class I, EpCAM, ICAM-1, plakoglobin, Ki-67,p120 and uPA-receptor/CD87 (Pantel, J. Natl. Canc. Inst. 91, 1113–1124(1999)).

The here described method has potential for the study of gene expressionby rare cells in many other fields (as shown hereinbelow; for example,in the investigation of human restenotic tissue). For instance, theinvestigation of spatially and temporally regulated gene expression inembryogenesis and the analysis of stem cells and differentiated cells inadult tissues could be performed. Single cell analysis would greatlyadvance the understanding of a typical proliferation, metaplasia,pre-neoplastic lesions and carcinomata in situ.

A synopsis of genomic aberrations and the expression profiles of thesame cell may reveal the contingencies of different genotypes andphenotypes within a tumor cell population.

High-dose chemotherapy, surgery, and anti-angiogenic therapy approachescan target rapidly dividing cells and large tumor masses but areineffective in the elimination of remnant cells leading to minimalresidual disease. Adjuvant therapies, like antibody-based approachesRiethmuller, J. Clin. Oncol., 16, 1788–1794 (1998), are still based onprotein targets identified on the primary tumor. The here shown approachprovides now an opportunity to discover targets for minimal residualdisease by analyzing the micrometastatic cells directly.

EXAMPLE V Aberrant Gene Expression in Human Restenotic Tissue

The above described method was furthermore employed to detectdifferentially expressed genes in human restenotic tissue.

A high rate of restenosis is significantly limiting the success ofpercutaneous transluminal coronary angioplasty with subsequent stentimplantation as a frequent treatment of coronary atheroscleroticdisease. Although several cellular and molecular mechanisms have beenidentified in the development of in-stent restenosis, specific targetsfor an effective therapeutic prevention of restenosis are still scarce.In this study differentially expressed genes in microscopic atherectomyspecimen from human in-stent restenosis were identified.Immunohistochemistry showed that the restenotic material consistedmainly of smooth muscle cells (SMC) with rare infiltrates of mononuclearcells. cDNA samples prepared from restenotic specimen (n=10) and, ascontrol, from intima and media of healthy muscular arteries (n=10) wereamplified using a novel polymerase chain reaction protocol andhybridized to cDNA arrays for the identification of differentiallyexpressed genes. Expression of desmin and mammary-derived growthinhibitor was downregulated, whereas expression of FK506-binding protein12 (FKBP12), thrombospondin-1, prostaglandin G/H synthase-1, and the70-kDa heat shock protein B was found to be upregulated with highstatistical significance in human neointima. Using immunohistochemistry,FKBP12, a negative regulator of TGF-β signaling, was also upregulated atthe protein level in neointima providing a rationale for the therapeuticeffect of the FKBP12 ligand rapamycin in the treatment of a porcinerestenosis model.

To gain further insight into transcriptional and signaling eventsgoverning smooth muscle cell migration, proliferation and synthesis ofextracellular matrix, differential gene expression screening wasemployed using cDNA array technology with probes generated frommicroscopic specimen of human restenotic tissue. The power of thistechnology is the ability to simultaneously study in one sample theexpression of thousands of genes (Kurian, (1999) J Pathol 187:267–271).A previous hurdle of using this method was the need for micrograms ofmRNA or cRNA from samples usually composed of 10⁶–10⁷ cells. Here, thenovel technology, as described hereinabove, was employed. This allowedthe generation of representative cDNA amplificates from a single cell ora low number of cells in quantities sufficient for comprehensive cDNAarray hybridization.

10 specimen of each neointimal and quiescent media for the expression of2,435 genes of known function. While the expression of house-keepinggenes was largely comparable between normal and restenotic tissue closeto 10 percent of studied genes showed an increased or decreased level ofexpression. In the present study, it was focused on selected genes thathave previously been associated with restenosis. Desmin andmammary-derived growth factor inhibitor (MDGI) expression wasselectively downregulated while the expression of prostaglandin G/Hsynthase-1 (COX-1), thrombospondin-1 (TSP-1), heat-shock protein-70 B(hsp70B) and FK506-binding protein 12 (FKBP12) was found to beupregulated in human neointima hyperplasia. These findings were allconfirmed by gene-specific PCR. To study the significance of increasedgene expression in neointima, it was investigated whether increased mRNAlevels find their reflection in an increased protein level. Asexemplified with FKBP12 using immunohistochemistry, it was indeed founda robust overexpression of this regulator of TGF-β signaling inrestenotic tissue. This study shows that cDNA array technology can beused to reliably identify differentially expressed genes in healthy anddiseased human tissue even if only very small amounts of material areavailable.

The in-stent restenosis study group consisted of 13 patients whounderwent separate atherectomy procedures by Helix cutter deviceartherectomy (X-sizer, Endicor) within the renarrowed stent between 4–23month after primary stent implantation. All patients gave informedconsent to the procedure and received 15,000 units heparin before theintervention followed by intravenous heparin infusion, 1,000 units/h forthe first 12 h after sheat removal as standard therapy. All patientsreceived aspirin, 500 mg intravenously, before catherisation, andpostinterventional antithrombotic therapy consisted of ticlopidine (250mg bds) and aspirin (100 mg bds) throughout the study.

Sample Preparation was Carried Out as Follows:

Atherectomy specimen were immediately frozen in liquid nitrogen afterdebulking of the lesion, and kept in liquid nitrogen until mRNApreparation was performed as described. For histology andimmunhistochemistry of the in-stent restenotic tissue from coronaryarteries (n=3), the samples were fixed in 4% paraformaldehyd andembedded in paraffin as described.

The control group consisted of 5 specimen of muscular arteries of thegastrointestinal tract from five different patients and 5 specimen fromcoronary arteries from three different patients who underwent hearttransplantation. The control specimen were immediately frozen in liquidnitrogen. Prior to mRNA preparation, media and intima of the controlarteries were prepared and examined for atherosclerotic changes byimmunhistochemistry. If there were no atherosclerotic changes of thevessel morphology, the specimen (approx. 1×1 mm) were used as healthycontrol samples and mRNA and cDNA preparation was performed asdescribed.

For immunohistochemistry of FKBP12, neointima specimen of carotidrestenotic arteries (n=2) were obtained by atherectomy and immediatelyfrozen in liquid nitrogen after removal. Three 3 μm serial frozensections of the samples were mounted onto DAKO ChemMate™ Capillary GapMicroscope slides (100 μm).

mRNA Preparation and amplified cDNA was carried out as follows:

Specimen of quiescent vessels or in-stent restenotic tissue werequick-frozen and kept in liquid nitrogen until mRNA preparation and cDNAsynthesis was performed. Frozen tissue was ground in liquid nitrogen andthe frozen powder dissolved in Lysis/Binding buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 10 mM EDTA, pH 8.0, 1% LiDS, 5 mM dithiothreitol(DTT)) and homogenized until complete lysis was obtained. The lysate wascentrifuged for 5 mm at 10, 000 g at 4° to remove cell debris. mRNA wasprepared using the Dynbeads® mRNA Direct Kit™ (Dynal, Germany) followingthe manufacture's recommendation. Briefly, lysate was added to 50 μL ofpre-washed Dynabeads Oligo (dT)₂₅ per sample and mRNA was annealed byrotating on a mixer for 30 min at 4° C. Supernatant was removed andDynabeads Oligo (dT)₂₅/mRNA complex was washed twice with washing buffercontaining Igepal (50 mM Tris-HCl, pH 8.0, 75 mM KCl, 10 mM DTT, 025%Igepal), and once with washing buffer containing Tween-20 (50 mMTris-HCl, pH 8.0, 75 mM KCl, 10 mM DTT, 0.5% Tween-20).

cDNA was amplified by PCR using the procedure as described hereinabove.First-strand cDNA synthesis was performed as solid-phase cDNA synthesis.Random priming with hexanucleotide primers was used for reversetranscription reaction. mRNAs were each reversely transcribed in a 20 μLreaction volume containing 1× First Strand Buffer (Gibco), 0.01 M DTT(Gibco), 0.25% Igepal, 50 μM CFL5c-Primer (SEQ ID NO:8) [5′-(CCC)₅ GTCTAG A (NNN)₂-3′], 0.5 mM dNTPs each (MBI Fermentas) and 200 USuperscript II (Gibco), and incubated at 44° C. for 45 mm. A subsequenttailing reaction was performed in a reaction volume of 10 μL containing4 mM MgCl₂, 0.1 mM DTT, 0.2 mM dGTP, 10 mM KH₁₂PO₁₄ and 10 U of terminaldeoxynucleotide transferase (MBI Fermentas). The mixture was incubatedfor 24 mm at 37° C.

cDNA was amplified by PCR in a reaction volume of 50 μL containing 1×buffer 1 (Expand™ Long Template PCR Kit, Boehringer Mannheim), 3%deionized formamide, 1,2 μM CP2-Primer (SEQ ID NO:14) [5′-TCA GAA TTCATG (CCC)₅-3′], 350 μM dNTP and 4.5 U DNA-Polymerase-Mix (Expand™ LongTemplate PCR Kit, Roche Diagnostics, Mannhein). PCR reaction wasperformed for 20 cycles with the following cycle parameters: 94° C. for15 sec, 65° C. for 0:30 mm 68° C. for 2 mm; for another 20 cycles with:94° C. for 15 sec, 65° C. for 30 sec, 68° C. for 2:30+0:10/cycle mm; 68°C. 7 mm; 4° C. forever.

25 ng of each cDNA was labeled with Digoxigenin-11-dUTP (Dig-dUTP)(Roche Diagnostics) during PCR. PCR was performed in a 50 μL reactionwith 1× Puffer 1, 120 μM CP2 primer, 3% deionized formamide, 300 μMdTTP, 350 μM dATP, 350 μM dGTP, 350 μM dCTP, 50 μM Dig-dUTP, 4.5 UDNA-Polymerase-Mix. Cycle parameters were: one cycle: 94° C. for 2 mm;15 cycles: 94° C. for 15 sec, 63° C. for 15 sec, 68° C. for 2 mm; 10cycles: 94° C. for 15 sec, 68° C. for 3 min+5 sec/cycle; one cycle: 68°C., 7 min, 4° C. forever.

Hybridization of Clontech cDNA Arrays with dUTP-labeled cDNA Probes wascarried out as follows:

cDNA arrays were prehybridized in DigEASYHyb solution (RocheDiagnostics) containing 50 mg/L DNAsel (Roche Diagnostics) digestedgenomic E. coli DNA, 50 mg/L pBluescript plasmid DNA and 15 mg/L herringsperm DNA (Life Technologies) for 12 h at 44° C. to reduce background byblocking non-specific nucleic acid-binding sites on the membrane.Hybridization solution was hybridized to commercially available cDNAarrays with selected genes relevant for cancer, cardiovascular andstress response (Clontech). Each cDNA template was denatured and addedto the prehybridization solution at a concentration of 5 μg/mlDig-dUTP-labeled cDNA. Hybridization was performed for 48 hours at 44°C.

Blots were subsequently rinsed once in 2×SSC/0.1% SDS and once in1×SSC/0.1% SDS at 68° C. followed by washing for 15 min once in0.5×SSC/0.1% SDS and twice for 30 min in 0.1×SSC/0.1% SDS at 68° C. Fordetection of Dig-labeled cDNA hybridized to the array, the DigLuminescent Detection Kit (Boehringer, Mannheim) was used as describedin the user manual. For detection of the chemiluminescence signal,arrays were exposed to chemiluminescence films for 30 min at roomtemperature. Quantification of array data was performed by scanning ofthe films and analysis with array vision software (Imaging ResearchInc., St. Catharines, Canada). Background was subtracted and signalswere normalized to the nine housekeeping genes present on each filter,whereby the average of the housekeeping gene expression signals was setto 1 and the background set to 0. In a pilot study, six clones enrichedin one of the two probes were further analyzed by RT-PCR.

Results of the experimental studies are reported as mean expressionvalues of the ten examined specimen of the study or control group.Differences between the two patient groups were analyzed byWilcoxon-test (SPSS version 8.0). A p-value less than 0.03 was regardedas significant.

A selection of differential hybridization signals were confirmed by PCRusing gene-specific primers. PCR reactions were performed using 2.5 ngof each cDNA in 25 μl reaction containing 1×PCR buffer (Sigma), 200 μMdNTPs, 0.1 μM of each primer and 0.75 U Taq Polymerase (Sigma). Thefollowing primers were used: desmin, (SEQ ID NO: 17) 5′-ACG ATT CCC TGATGA GGC AG-3′ and (SEQ ID NO:18) 5′-CCA TCT TCA CGT TGA GCA GG-3′;thrombospondin-1, (SEQ ID NO:19) 5′-CTG AGA CGC CAT CTG TAG GCG GTG -3′and (SEQ ID NO:20) 5′-GTC TTT GGC TAC CAG TCC AGC AGC-5′;mammary-derived growth inhibitor, (SEQ ID NO:21) 5′-AAG AGA CCA CAC TTGTGC GG-3′ and (SEQ ID NO:22) 5′-AAT GTG GTG CTG AGT CGA GG-5′;prostaglandin G/H synthase-1, (SEQ ID NO:23) 5′-CGG TGT CCA GTT CCA ATACC-3′ and (SEQ ID NO:24) 5′-CCC CAT AGT CCA CCA ACA TG-3′; FKBP12, (SEQID NO:25) 5′-ATG CCA CTC TCG TCT TCG AT-3′ and (SEQ ID NO:26) 5′-GGA ACATCA GGA AAA GCT CC-3′; heat shock protein 70B, (SEQ ID NO:27) 5′-TAC AAGGCT GAG GAT GAG GC-3′ and (SEQ ID NO:28) 5′-CTT CCC GAC ACT TGT CTTGC-3′, and β-actin, (SEQ ID NO:29) 5′-CTA CGT CGC CCT GGA CTT CGA GC-5′and (SEQ ID NO:30) 5′-GAT GGA GCC GCC GAT CCA CAC GG-3′. PCR productswere subjected to electrophoresis on a 2% agarose gel containingethidium bromide (0.5 μg/ml agarose solution) in TAE buffer (20 mMTris/HCl, 10 mM acetic acid, 1 mM EDTA).

Immunohistochemistry was carried out as follows:

Immunohistochemistry for cell typing was performed on paraffin-embeddedsections of three neointima specimen from coronary in-stent restenosisand, for detection of FKBP12, on frozen sections of four neointimaspecimen from carotid restenosis. Three μm serial sections were mountedonto DAKO ChemMate™ Capillary Gap Microscope slides (100 μm) baked at65° C. overnight, deparaffinized and dehydrated according to standardprotocols. For antigen retrieval, specimens were boiled 4 min in apressure cooker in citrate buffer (10 mM, pH 6.0). Endogenous peroxidasewas blocked by 1% H₂O₂/methanol for 15 minutes. Unspecific binding ofthe primary antibody was reduced by preincubation of the slides with 4%dried skim milk in Antibody Diluent (DAKO, Denmark). Immunostaining wasperformed by the streptavidin-peroxidase technique using the ChemMateDetection Kit HRP/Red Rabbit/Mouse (DAKO, Denmark) according to themanufacturer's description. The procedures were carried out in a DAKOTechMate™ 500 Plus automated staining system. Primary antibodies againstsmooth muscle actin (M0635, DAKO, Denmark; 1:300), CD3 (A0452, DAKO,Denmark; 1:80), MAC387 (E026, Camon, Germany; 1:20) and FKBP12 (SA-218,Biomol, Germany, 1:20) were diluted in Antibody Diluent and incubatedfor 1 h at room temperature. After nuclear counterstaining withhematoxylin, the slides were dehydrated and coverslipped with Pertex(Medite, Germany).

For FKBP12 immunhistochemistry, 3 μm frozen, serial sections of theneointima specimen from carotid restnosis were mounted onto DAKOChemMate™ Capillary Gap Microscope slides (100 μm).

The following results were obtained:

(a) The Cellular Composition of Debulked In-stent Restenotic Material

-   -   Representative samples obtained from x-sizer treatment of a        neointimal hyperplasia were analyzed by immunhistochemistry in        order to determine its cellular composition. The restenotic        tissue analyzed was removed by x-sizer debulking from coronary        arteries more than two month after PTCA and stent implantation.        The amount of tissue generated by this procedure was very low        containing an estimated 300–10000 cells. FIG. 7A shows an        E.-van-Giesson staining of a section cut from a small sample of        debulked restenotic material. With this staining procedure,        collagen fibers stain red, fibrin stains yellow and cytoplasm of        smooth muscle cells stains dark-yellow-brown. The majority of        the volume of debulked material was composed of loose        extracellular matrix-like collagen fibers stained in light red.        Yellow fibrin staining was barely detectable. Cells with        spindle-shaped nuclei and a yellow/brown-stained cytoplasm were        frequent. Their identity as smooth muscle cells and their high        abundance in restenotic material was supported by immunostaining        with an antibody against smooth muscle α-actin (FIG. 7B). There,        the staining pattern of a section from an entire specimen as        used for gene expression analysis is shown. As described below,        such samples also gave raise to a strong smooth muscle-specific        α-actin mRNA signal (see FIG. 8). These results support findings        from previous studies (Komatsu, (1998), Circulation 98:224–233;        Strauss (1992), J. Am. Coll. Cardiol. 20:1465–1473; Kearney        (1997), Circulation 95:1998–2002) demonstrating that the main        cell type found in neointima is derived from smooth muscle        cells. As described in the literature, mononuclear infiltrates        in some areas of debulked restenotic tissue specimen could also        be identified (data not shown). These infiltrates consisted        mainly of macrophages and to a lesser degree of t-lymphocytes.        No b-lymphocytes were detectable in the restenotic tissue by        using an antibody against CD20 for immunhistochemical staining        (data not shown).        (b) Expression of Specific Genes in Microscopic Human Tissue        Samples    -   In order to optimally preserve the in situ mRNA levels,        restenotic and control specimen were immediately frozen after        harvest in liquid nitrogen and carefully lyzed as described        hereinabove. After PCR amplification of the synthesized cDNA the        amount of the amplified cDNA was measured by a dot blot assay        and found to be between 200–300 ng/μl. The quality of every        amplified cDNA sample was tested by gene-specific PCR using        primers detecting cDNAs for β-actin, smooth muscle cell α-actin        and the ubiquitous elongation factor EF-1α. FIG. 8 shows a        representative result with material from patient B and control        media from donor b. In both specimen, PCR signals of the correct        size from house-keeping genes β-actin and EF-1α were detectable        in equivalent amounts (compare lanes 1 and 2 with lanes 4 and        5). Additionally, α-actin signals as marker for smooth muscle        cells was obtained from each specimen (lanes 3 and 6). These        results show that mRNA preparation, cDNA synthesis and PCR        amplification of cDNA is feasible with microscopic human        restenosis samples.        (c) Comparative Gene Expression Profiling Using Microscopic        Human Tissue Samples    -   To identify differentially expressed mRNAs in restenotic versus        healthy specimen, the cDNAs was labeled during PCR amplification        with digoxigenin-labeled dUTP as described hereinabove. This        label allows for a highly sensitive, chemiluminescence-based        detection of hybridization signals of cDNA arrays on        photographic films. The nylon filters with cDNA arrays were        pre-hybridized with DNAsel-digested genomic E. coli DNA and with        DNAsel-digested pBluescript plasmid DNA. This procedure was        employed to maximally reduce non-specific DNA binding to the        array. Each labeled probe was hybridized to three different        commercial cDNA arrays which allowed for the expression analysis        of a total of 2,435 known genes. FIG. 9 shows a representative        hybridization pattern obtained with one array using probes        prepared from restenotic tissue of patient B (panel A) and media        of donor b (panel B). Consistent with the gene-specific analysis        shown in FIG. 8, comparable hybridization signals were obtained        with the positive control of human genomic cDNA spotted on the        right and bottom lanes of the array and with cDNA spots of        various housekeeping genes (see for instance, spots D). If a        biological specimen was omitted from cDNA synthesis and PCR        amplification reactions almost no hybridization signals were        obtained (FIG. 9, panel C), showing that hybridization signals        were almost exclusively derived from added samples and not from        DNA contaminations in reagents or materials used.    -   Visual inspection of the hybridization patterns readily        identified a number of signals that are different between        healthy and diseased tissue (for instance signals A, B and C in        FIGS. 9A and B). Samples from restenotic tissues consistently        gave more signals than control tissues. Hybridization signals        obtained from the use of three different cDNA arrays with 10        restenosis patient samples and 10 normal media samples were        quantitated by densitometric analysis of photographic films and        the data electronically compiled and further analyzed for        statistics. Expression levels for 53 out of 2,435 genes is shown        in FIG. 10 whereby one grey value corresponds to the signal        intensity as shown in the figure legend. A considerable        variation of gene expression is evident for most genes shown        which may reflect genetic and physiological differences of        patients and donors. For further analysis and verification by        gene-specific PCR, only genes were considered that showed a        differential expression with a statistical difference of at        least p=0.03 by the Wilcoxon Test. Six such genes are        highlighted in the list (FIG. 10). A total of 224 genes out of        2435 known genes was found to be differentially regulated in        neointima with high statistical significance. Their        comprehensive in-depth analysis will be published elsewhere.        Indicative for a comparable sample quality, eight housekeeping        genes showed very similar hybridization signal intensities with        all 20 samples (FIG. 10, bottom).        (d) Validation of cDNA Array Data by Gene-specific PCR    -   Out of the list depicted in FIG. 10, six differentially        regulated genes and one housekeeping gene were selected for        validation of hybridization signals through PCR using        gene-specific primers. All PCR signals obtained had the        predicted size. In support of an equal quality of samples, the        β-actin signal (bottom) showed a very similar intensity with all        20 samples. By comparing gene-specific PCR signals (FIG. 11)        with hybridization signals obtained from cDNA arrays (FIG. 11)        it was found that 135 out of 140 signals matched with respect to        intensity. This corresponds to a 96% fidelity of hybridization        signals from cDNA arrays showing that the here employed gene        expression profiling approach is comparable with respect to        quality and sensitivity to gene-specific PCR.        (e) Aberrant Gene Expression in Human Restenotic Tissue    -   Desmin, a mesenchymal marker, was found strongly expressed in        the control media, whereas only weak signals were found in the        restenotic specimen (FIGS. 10 and 11). Desmin is a marker for        SMCs that is highly expressed in quiescent, differentiated SMCs.        Its expression is reduced in de-differentiated, proliferating        SMCs, e.g., in SMCs of atherosclerotic plaques (Ueda (1991),        Circulation 83:1327–1332). Downregulation of desmin in        restenotic tissue implies that the spindle-shaped cells in the        restenotic material are de-differentiated, proliferating SMCs.        Inversely, TSP-1, an extracellular matrix protein, that is        important in TGF-β activation and SMC migration and        proliferation (Yehualaeshet (1999), Am J Pathol 155:841–851;        Scott (1988), Biochem. Biophys. Res. Commun. 150:278–286), is        markedly upregulated in the majority of neointimal specimen        versus the control samples. The COX-1, stress-induced hsp70B and        the ubiquitously expressed FKBP12 genes were significantly        upregulated in almost all neointimal hyperplasia and barely, if        at all, expressed in control specimen (FIGS. 10 and 11). The        tumor suppressor MDGI was strongly expressed in quiescent smooth        muscle whereas little expression was found in a few neointima        hyperplasia samples. None of the restenotic lesions expressed        desmin (0/0) compared to 100% of controls (10/10), only 30%        (3/10) of the neointimal specimen expressed MDGI very slightly,        whereas it was highly expressed in 8/10 (80%) of the controls.        Otherwise, TSP-1 (7/10), COX-1 (9/10), hsp70B (8/10) and FKBP12        (10/10) were significantly upregulated in neointimal versus        control specimen (TSP-1 [0/10], hsp70B [0/10], COX-1 [0/10],        FKBP12 [1/10]).        (f) FKBP12 Protein Expression is Upregulated in Human Restenotic        Tissue    -   Upregulation of mRNA levels does not stringently indicate an        increased level of protein. Among the genes that were found to        be upregulated in human neointima, FKBP12 is particularly        interesting since it is a regulator of TGF-β signaling and        target for the drugs FK506 and rapamycin. A therapeutic effect        of rapamycin in rodent models (Gallo (1999), Circulation        99:2164–2170) of restenosis is poorly understood but may be        related to changes in the expression level of FKBP12. Using an        antibody specific for FKBP12, human restenotic tissue from        carotid restenosis (n=3) was analyzed and control tissue (n=3)        for the expression of the protein. As shown in FIG. 12, an        increase in FKBP12 protein in the cytoplasm of SMCs from        restenotic lesions as identified by their spindle-shaped nuclei        was detected (FIGS. 12B and D). Whereas no FKBP12 was detectable        in control SMCs of healthy media (FIG. 12C), a distinct staining        in SMCs of neointima was found (FIG. 12D). Interestingly,        especially smooth muscle cells lying in the border zone between        neointima and healthy media of restenotic vessels expressed high        levels of the FKBP12 protein (FIG. 11B).

EXAMPLE VI Characterization of the Transcriptome of Human RestenoticTissue

The expression of 2,435 genes of known function (see Example V) wasinvestigated in atherectomy specimen of 10 patients with in-stentrestenosis, blood cells of 10 patients, normal coronary artery specimenof 11 donors, and cultured human coronary artery smooth muscle cells.224 genes that were differentially expressed with high statisticalsignificance (p<0.03) between neointima and control tissue which couldbe grouped as follows: (1) genes only expressed in neointima; (2) genesexpressed in both neointima and proliferating smooth muscle cells; (3)genes expressed in both neointima and blood samples; and (4) genesexpressed in control tissue but barely in neointima. The transcriptomeof human neointima showed significant changes related to proliferation,apoptosis, inflammation, cytoskeletal reorganization and tissueremodeling. Furthermore, in neointima 32 upregulated genes wereidentified that are related to interferon-γ signaling.

In the present study, 10 specimen of neointimal and 11 specimen ofquiescent intima/media for the expression of 2,435 human genes of knownfunction were analyzed. While the expression of housekeeping genes waslargely comparable between normal and restenotic tissue, an impressivenumber of genes (n=224) showed an increased or decreased level ofexpression. The gene expression pattern in neointima showed theanticipated proliferative response with induction of genes mainlyexpressed in G1/S phase, changes of the smooth muscle phenotype fromcontractile to synthetic SMCs and changes in synthesis of extracellularmatrix proteins. Additionally, a pro-inflammatory expression patterncharacterized by the presence of markers for macrophages and Tlymphocytes and by the expression of numerous genes with known functionsin the cellular response to IFN-γ were observed. The IRF-1 protein, apivotal transcription factor in IFN-γ signaling, was found overexpressedin SMCs of human neointima.

The clinical characteristics of the patients of the study group of thisExample are presented in Table 7.

TABLE 7 Clinical Data of 13 Patients Interval/ Stent/ Interval Hyper-Arterial Stent Poststent Stent/ cholester- Hyper- Diabetes Multivesselfamilial Patient Age, y Sex Indication for Stent Site RestenosisDebulking Smoker olemia tension mellitus disease risk 1 77 m AMI RCA 5 m11 m  − + + + + − 2 62 m SAP LAD 6 m 19 m  − + + − − + 3 57 m ISAP ACVB4 m 10 m  + − + + + − 7 4 68 m failed Bypass ACVB 4 m 4 m − + + − + − 145 80 m AMI LAD 7 m 7 m − + + − + − 6 67 m Restenosis RCA 12 m  23 m − + + − + − 7 44 f Restenosis after PTCA RCA 3 m 8 m − + 7 − − − 8 75 mRestenosis after PTCA RCA 6 m 6 m − + − − + − 9 86 m Restenosis afterPTCA RCA 5 m 5 m − + + − − − 10 44 m Restenosis after PTCA LAD 6 m 6m + + + − − + 11 76 m AMI LAD 6 m 6 m − + + − − − 12 46 m Restenosisafter PTCA LAD 5 m 5 m − + + − − + 13 69 m Restenosis after PTCA LAD 4 m16 m  − + + − + −

All atherectomy specimen were immediately frozen in liquid nitrogenafter debulking of the lesion, and kept in liquid nitrogen until mRNApreparation was performed as described above.

The control group consisted of 5 specimen of muscular arteries of theintestine from five patients and 6 specimen from coronary arteries fromthree patients who underwent heart transplantation. The control specimenwere immediately frozen in liquid nitrogen. Prior to mRNA preparation,media and intima of the arteries were prepared. A small piece of thespecimen (approx. 1 mm³) was immediately lysed, whereas the rest washistologically examined for atherosclerotic changes. If there were noatherosclerotic changes of vessel morphology detectable, the specimenwere used as “healthy” control samples and mRNA and cDNA preparation wasperformed as described.

The neointimal tissue of carotid (n=3) and femoralis (n=3) arteries wasgenerated by atherectomy within the restenosis and immediately frozenafter removal in liquid nitrogen. For histologic evaluation andimmunohistochemistry of the in-stent restenotic tissue from coronaryarteries (n=3) and of the neointima of restenotic peripheral arteries(n=6), the samples were fixed in 4% paraformaldehyd and embedded inparaffin as described.

Blood samples were obtained immediately after revascularization of therestenotic vessel. Eight ml blood samples were collected into 35 ml ofTriReagent Blood (MBI Fermentas, Germany) and subsequently frozen at−80° C. until RNA preparation was performed as described in themanufacture's protocol. 1 μg of total RNA of blood cells were dissolvedin 1000 μL Lysis/Binding buffer and mRNA and cDNA synthesis was preparedas described above.

Cell Culture was carried out as follows:

Primary human coronary artery smooth muscle cells (CASMCs) were obtainedfrom CellSystems (St. Kathrinen, Germany) and were grown in SmoothMuscle Cell Growth Medium (CellSystems, St. Kathrinen, Germany)containing 5% fetal calf serum (CellSystems, St. Kathrinen, Germany) at37° C. in a humidified atmosphere of 5% CO₂. CASMCs were used inexperiments between passages 2 and 4. For cDNA synthesis ofproliferating CASMCs were washed three times with ice-coldphosphate-buffered saline and 1×10⁴ cells were subsequently lysed in1000 μL Lysis/Binding puffer before mRNA was prepared as describedabove.

Determination of Gene Expression Patterns was carried out as follows:

Sample mRNA preparation, cDNA synthesis, PCR amplification and probelabeling, cDNA array hybridization and data analysis were performed asdescribed hereinabove, in particular in Example V. The obtained cDNAprobes were hybridized to Human 1.2, Cancer 1.2, Cardiovascular andStress cDNA arrays (Clontech, Heidelberg, Germany) with a total of 2,435genes of known function. There was an approximately 20% redundancy ofgenes among cDNA arrays. For analysis of microscopic human tissuesamples down to a single cell level the here described new method ofcDNA synthesis and PCR amplification was used (see Examples I to V).

Quantification of array data was performed by scanning of the films andanalysis with array vision software (Imaging Research Inc., St.Catharines, Canada). Background was subtracted and signals werenormalized to the nine housekeeping genes present on each filter,whereby the average of the housekeeping gene expression signals was setto 1 and the background set to 0. For the logarithmic presentation shownin FIG. 1, values were multiplied by 1000. A mean value ≧0,05 in theaverage of all samples in one group was regarded as a positive signal.Differences in the mean expression level by a factor ≧2.5-fold betweenthe study and the control group were further statistically analyzed.

Results of the experimental analysis are given as mean expression valuesof the ten examined specimen of the study group or the eleven examinedspecimen of the control group. Differences between the patient and donorgroups were analyzed by the Wilcoxon-test (SPSS version 8.0). Genes wereonly considered to be differentially expressed between the two groups iftheir p-values in the Wilcoxon test were <0.03, and if a differentialexpression was observed in at least 5 out of 10 samples within one studygroup, while there was 0 out of 10 within the other group; or at least 7out of 10 samples within one group, while there were maximally 3 out of10 within the other group.

Immunhistochemistry was carried out as follows:

Immunhistochemistry was performed on paraffin-embedded sections from 3neointima specimen from coronary in-stent restenosis, 3 neointimaspecimen from A. femoralis and 3 neointima specimen from carotidneointima specimen. Three μm serial sections were mounted onto DAKOChemMate™ M Capillary Gap Microscope slides (100 μm), baked at 65° C.overnight, deparaffinized and dehydrated according to routine protocols.For antigen retrieval, specimen were boiled 4 minutes in a pressurecooker in citrate buffer (10 mMol, pH 6.0). Endogenous peroxidase wasblocked by 1% H₂O₂/methanol for 15 minutes. Unspecific binding of theprimary antibodies was reduced by preincubation of the slides with 4%dried skim milk in Antibody Diluent (DAKO, Denmark). Immunostaining wasperformed by the streptavidin-peroxidase technique using the DakoChemMate Detection Kit HRP/Red Rabbit/Mouse (DAKO Denmark) according tothe manufacturers description. The procedures were carried out in a DAKOTechMate™ 500 plus automated staining system. Primary antibodies againstsmooth muscle actin (M0635, DAKO, Denmark; 1:300), CD3 (A0452, DAKO,Denmark; 1:80), MAC387 (E026, Camon, Germany; 1:20) and IRF-1 (sc-497,Santa Cruz, U.S.A.) were diluted in Antibody Diluent and incubated for 1h at room temperature. After nuclear counterstaining with hematoxylin,slides were dehydrated and coverslipped with Pertex (Medite, Germany).

The following results were obtained:

(a) Differential Gene Expression in Human Neointima

-   -   A total of 1,186 genes (48.7%) out of 2,435 yielded detectable        hybridization signals on cDNA arrays with neointima and control        samples over a 20-fold range of expression level (FIG. 13A)        Thereof 352 genes (14.5%) appeared to be differentially        expressed by a factor >2.5-fold between restenotic and control        samples. However, expression levels considerably varied among        individual samples (see, e.g., FIG. 15). Therefore, a        statistical analysis was employed to identify those genes that        are differentially expressed between study and control groups        with high significance (see Methods). This way, 224 genes (9.6%)        were identified that were differentially expressed by a factor        of at least 2.5-fold between the restenosis study group and the        control group with a significance in the Wilcoxon test of        p<0.03. 167 (75%) genes thereof were found overexpressed and 56        genes (25%) underexpressed in the restenosis study group        compared to the control group (FIG. 13B).    -   In addition to the statistical significance, the validity of        expression data was supported by a 20% redundancy of cDNA        elements on the four arrays used. This way, a substantial number        of hybridization signals was determined in duplicate or        triplicate in independent hybridization experiments. Four        examples of duplicate determinations are shown in FIG. 16 (top)        which all showed a high degree of reproducibility. As a further        validation of hybridization signals, 38 of the differentially        expressed genes were selected for PCR analysis of cDNA samples        using gene-specific primers. Hybridization signals for 35 (92%)        out of 38 genes could be verified by gene-specific PCR yielding        signals of the predicted size and relative quantity (data not        shown). These data shows that the employed cDNA array approach        is comparable with respect to quality and sensitivity to        gene-specific PCR. Lastly, among the 224 aberrantly expressed        genes in neointima 112 have previously been described in the        literature as being expressed in neointima, SMCs, fibroblasts,        endothelial cells or mesenchym (FIG. 14 marked with ‘#’).    -   With respect to neointima expression, the 224 aberrantly        regulated genes fell into four subgroups (FIG. 14). Group I        lists 62 genes that were overexpressed in neointima and not        highly or detectably expressed in control vessels, CASMCs or        blood cells (FIG. 14A). In group II, 43 genes are listed that        are similarly expressed in neointima and CASMCs, suggesting that        this gene cluster in neointima was contributed by proliferating        SMCs (FIG. 14B). In group III, 62 genes are listed that are        similarly expressed in neointima and blood cells, suggesting        that this gene cluster was contributed to that of neointima by        infiltrated blood cells (FIG. 14C). This notion is supported by        the expression in group III of the largest number of genes        related to inflammation in all four groups. Lastly, in group IV,        56 genes are listed that are downregulated in neointima compared        to the control group (FIG. 14D). In the following, the aberrant        expression of selected genes in neointima will be discussed in        the context of gene function.

In summary, the following differentially expressed genes have beendetected in human neointima:

GenBank SwissProt Gene Name Accession # Accession # 80-kDa nuclearcap-binding protein D32002 Q09161 activator 1 140-kDa subunit (A1140-kDa subunit); replication factor C large subunit; DNA-binding L14922P35251 protein PO-GA activator 1 37-kDa subunit; replication factor C37-kDa subunit (RFC37); RFC4 M87339 P35249 adenylate kinase isoenzyme 1(AK1); ATP-AMP transphosphorylase; myokinase J04809 P00568 adipocytefatty acid-binding protein 4 (FABP4; AFABP); adipocyte lipid-bindingprotein (ALBP) J02874 P15090 allograft inflammatory factor 1 (AIF1);ionized calcium-binding adapter molecule 1 U19713 P55008alpha-1-antitrypsin precursor; alpha-1 protease inhibitor;alpha-1-antiproteinase X02920 P01009 alpha-2-antiplasmin D00174 P08697alpha-2-macroglobulin precursor (alpha-2-M) M11313 P01023alpha-galactosidase A precursor; melibiase; alpha-D-galactosidegalactohydrolase X05790 P06280 amiloride-sensitive epithelial sodiumchannel beta subunit; nonvoltage-gated sodium channel 1 beta X87159P51168 subunit (SCNEB; beta NACH); SCNN1B angiotensinogen precursor(AGT) K02215 P01019 apolipoprotein E precursor (APOE) M12529 P02649atrial natriuretic peptide receptor B precursor (ANPB; NPRB); guanylatecyclase B (GCB) L13436 P20594 autosomal dominant polycystic kidneydisease II (PKD2) U50928 Q13563 B-cell-associated molecule CD40 X60592P25942 BCL-2 binding athanogene-1 (BAG-1); glucocorticoidreceptor-associated protein RAP46 S83171; Z35491 Q99933 BCL-2-relatedprotein A1 (BCL2A1); BFL1 protein; hemopoietic-specific early responseprotein; GRS U29680; Y09397 Q16548; protein Q99524 BIGH3 M77349 Q15582bikunin; hepatocyte growth factor activator inhibitor 2 U78095 O00271;O43291 brain glucose transporter 3 (GTR3) M20681 P11169 brain-specificpolypeptide PEP-19; brain-specific antigen PCP-4 U52969 P48539 Bruton'styrosine kinase (BTK); agammaglobulinaemia tyrosine kinase (ATK); B-cellprogenitor kinase U10087; X58957 Q06187 (BPK) C5a anaphylatoxin receptor(C5AR); CD88 antigen M62505 P21730 cadherin 16 (CDH16); KSP-cadherinAF016272 P75309 calcium & integrin-binding protein (CIB) U85611 Q99828carboxypeptidase H precursor (CPH); carboxypeptidase E (CPE); enkephalinconvertase; prohormone X51405 P16870 processing carboxypeptidasecarboxypeptidase N X14329 P15169 caspase-8 precursor (CASP8); ICE-likeapoptotic protease 5 (ICE-LAP5); MORT1-associated CED-3 U60520; Q14790;homolog (MACH); FADD-homologous ICE/CED-3-like protease (FADD-like ICE;FLICE); apoptotic U58143; Q15780 cysteine protease MCH-5 X98172; AF00962caveolin 3 AF043101 P56539 CBL-B U26710 Q13191 CDC42 homolog; G25KGTP-binding protein (brain isoform + placental isoform) M35543 + M57298P21181 + P25763 cell surface adhesion glycoproteins LFA-1/CR3/p150,95beta-subunit precursor; LYAM1; integrin beta 2 M15395 P05107; (ITGB2);CD18 antigen; complement receptor C3 beta subunit Q16418 cell surfaceglycoprotein mac-1 alpha subunit precursor; CD11B antigen; leukocyteadhesion receptor J04145 P11215 MO1; integrin alpha M (ITGAM);neutrophil adherence receptor alpha M subunit; CR3A cell surfaceglycoprotein MUC18; melanoma-associated antigen A32; CD146 antigen;melanoma M28882 P43121 adhesion molecule C-fgr proto-oncogene (p55-FGR);SRC2 M19722 P09769 c-fos proto-oncogene; G0S7 protein K00650 P01100chemokine receptor-like 2; IL8-related receptor DRY12; flow-inducedendothelial G protein-coupled AF015257 Q99527; receptor (FEG1); Gprotein-coupled receptor GPR30; GPCR-BR) Q99981; O00143; Q13631 clone23815 (Soares library 1NIB from IMAGE consortium) U90916 nonecoagulation factor XII M11723 P00748 collagen 16 alpha 1 subunitprecursor (COL16A1) M92642 Q07092 collagen 18 alpha 1 subunit (COL18A1)L22548 P39060 collagen 6 alpha 1 subunit (COL6A1) X15880 P12109 collagen6 alpha 2 subunit (COL6A2) M34570 Q13909; Q13911 coronin-like proteinP57 D44497 P31146 c-src kinase (CSK); protein-tyrosine kinase cyl X59932P41240 cyclin-dependent kinase 4 inhibitor (CDK4I; CDKN2); p16-INK4;multiple tumor suppressor 1 (MTS1) L27211 P42771; Q15191cyclin-dependent kinase inhibitor 1 (CDKN1A); melanomadifferentiation-associated protein 6 (MDA6); U09579; L25610 P38936CDK-interacting protein 1 (CIP1); WAF1 cytidine deaminase (CDA) L27943P32320 death-associated protein 1 (DAP1) X76105 P51397 desmin (DES)U59167 P17661; Q15787 DNAX activation protein 12 AF019562 O43914dual-specificty A-kinase anchoring protein 1 X97335 Q92667 early growthresponse protein 1 (hEGR1); transcription factor ETR103; KROX24; zincfinger protein 225; X52541; M62829 P18146 AT225 early response proteinNAK1; TR3 orphan receptor L13740 P22736 endothelial differentiation gene1 (EDG1) M31210; P21453 AF022137 endothelin 2 (ET2) M65199 P20800 ephrinA receptor 4 precursor; tyrosine-protein kinase receptor sek; hek8L36645 P54764 epithelial discoidin domain receptor 1 precursor (EDDR1;DDR1); cell adhesion kinase (CAK); TRKE; X74979 RTK6 estradiol 17beta-dehydrogenase 1 M36263 P14061 estrogen-related receptor alphaX51416; Y00290 P11474 ets domain protein elk-3; NET; SRF accessoryprotein 2 (SAP2) Z36715 P41970 extracellular superoxide dismutaseprecursor (EC-SOD; SOD3) J02947 P08294 farnesyltransferase beta L10414P49356 FC-epsilon-receptor gamma subunit M33195 P30273 FK506-bindingprotein (FKBP; FKBP12); peptidyl-prolyl cis-trans isomerase (PPIASE);rotamase M34539; M80199; M80706; M92423; J05340; X55741; X52220 fli-1oncogene; ergB transcription factor M93255 Q01543 FMLP-related receptorI (FMLPRII); RMLP-related receptor I (RMLPRI) M76673 P25089 focaladhesion kinase 2 (FADK2; FAK2); cell adhesion kinase beta (CAKbeta);proline-rich tyrosine L49207 + U43522 + U33284 Q14289; kinase 2 (PYK2)Q16709; Q13475 frizzled-related FrzB (FRITZ) + FrzB precursor + frezzled(FRE) U91903 + U24163 + U68057 O00181 + Q92765 + Q99686 Gprotein-coupled receptor EDG4 AF011466 O43431 G1/S-specific cyclin D1(CCND1); cyclin PRAD1; bcl-1 oncogene X59798; M64349 P24385G1/S-specific cyclin D3 (CCND3) M92287 P30281 gamma-interferon-inducibleprotein; IP-30 J03909 P13284 GAP junction alpha-1 protein X52947 P17302glutathione-S-transferase (GST) homolog U90313 P78417 glycerol kinaseL13943 P32189 G-protein-coupled receptor HM74 D10923 P49019 granulocytecolony stimulating factor receptor precursor (GCSF-R); CD114 antigenM59818 Q99062 granulocyte-macrophage colony-stimulating factor receptoralpha (GM-CSFR-alpha); CSW116 antigen X17648 P15509 growth arrest &DNA-damage-inducible protein 45 beta (GADD45 beta) AF078077 none growtharrest & DNA-damage-inducible protein 45 gamma (GADD45 gamma) AF078078none growth factor receptor-bound protein 2 (GRB2) isoform; GRB3-3;SH2/SH3 adaptor GRB2; L29511; M96995 P29354 ASH protein + epidermalgrowth factor receptor-bound protein 2 (EGFRBP-GRB2) growth inhibitoryfactor; metallothionein-III (MT-III) D13365; M93311 P25713 GTP-bindingprotein ras associated with diabetes (RAD1) L24564 P55042 guaninenucleotide-binding protein G(Y) alpha 11 subunit (GNA11; GA11) M69013P29992; Q14350; O15109 heart fatty acid-binding protein 3 (FABP3;HFABP); muscle fatty acid-binding protein (MFABP); Y10255 P05413;mammary-derived growth inhibitor (MDGI) Q99957 heat shock 70-kDa protein6 (heat shock 70-kDa protein B) X51757; M11236 P48741 heat shock cognate71-kDa protein Y00371 P11142 heme oxygenase 1 (HO1); HSOXYGR X06985P09601 high mobility group protein (HMG-I) M23619 P17096 high-affinityinterleukin-8 receptor A (IL-8R A); IL-8 receptor type 1; CDW128 M68932P25024 high-affinity nerve growth factor receptor precursor; trk-1transforming tyrosine kinase protein; p140- X03541 P04629 TRKA;p68-trk-T3 oncoprotein histone H4 X67081 none HLA class IIhistocompability antigen alpha subunit precursor (MHC-alpha) M31525P06340 homeobox protein HOXB7; HOX2C; HHO.c1 M16937 P09629hormone-sensitive lipase Q05469 hydroxyacyl-CoA dehydrogenase;3-ketoacyl-CoA thiolase; enoyl-CoA hydratase beta subunit D16481 P55084IgG receptor FC large subunit P51 precursor (FCRN); neonatal FCreceptor; IgG FC fragment receptor U12255 P55899 transporter alpha chainIMP dehydrogenase 1 J05272 P20839 insulin receptor precursor (INSR)M10051; X02160 P06213 insulin-like growth factor binding protein 6precursor (IGF-binding protein 6; IGFBP6; IBP6) M62402 P24592insulin-like growth factor I receptor (IGF1R) X04434; M24599 P08069integrin alpha 3 (ITGA3); galactoprotein B3 (GAPB3); VLA3 alpha subunit;CD49C antigen M59911 P26006 integrin alpha 7B precursor (IGA7B) X74295Q13683 integrin alpha 8 (ITGA8) L36531 P53708 integrin beta 7 precursor(ITGB7) M62880; S80335 P26010 inter-alpha-trypsin inhibitor heavy chainH4 precursor (ITI heavy chain H4); plasma kallikrein-sensitive D38595Q14624 glycoprotein 120 (PK-120) intercellular adhesion molecule 2precursor (ICAM2); CD102 antigen X15606 P13598 intercellular adhesionmolecule 3 precursor (ICAM3); CDW50 antigen; ICAM-R X69711; X69819P32942 intercellular adhesion molecule-1 precursor (ICAM1); major grouprhinovirus receptor; CD54 antigen J03132 P05362 interferon regulatoryfactor 1 (IRF1) X14454 P10914 interferon regulatory factor 7 (IRF-7)U73036 Q92985 interferon-gamma (IFN-gamma) receptor beta subunitprecursor; IFN-gamma accessory factor 1 (AF1); U05875 P38484 IFN-gammatransducer 1 (IFNGT1) interferon-gamma receptor (IFNGR) A09781 noneinterferon-induced 56-kDa protein (IFI-56K) X03557 P09914interferon-inducible protein 9–27 J04164 P13164 interleukin-1 betaconvertase precursor (IL-1BC); IL-1 beta converting enzyme (ICE); p45;caspase-1 U13699; M87507; P29466 (CASP1) X65019 interleukin-1 receptortype II precursor (IL-1R2); IL-1R-beta X59770 P27930 interleukin-16(IL-16); lymphocyte chemoattractant factor (LCF) M90391 Q14005interleukin-2 receptor gamma subunit (IL-2R gamma; IL2RG); cytokinereceptor common gamma chain D11086 P31785 precursor; p64 interleukin-6receptor alpha subunit precursor (IL-6R-alpha; IL6R); CD126 antigenM20566; X12830 P08887 I-rel (RELB) M83221 Q01201 leukocyte IgG receptor(FC-gamma-R) J04162 P08637 lipoprotein-associated coagulation inhibitorJ03225 P10646 low affinity immunoglobulin gamma FC receptor II-Aprecursor (FC-gamma RII-A; FCRII-A; IgG FC M31932 P12318 receptor II-A);CD32 antigen low-density lipoprotein receptor-related protein LR11precursor Y08110 Q92673 L-selectin precursor; lymph node homing receptor(LNHR); leukocyte adhesion molecule 1 (LAM1) M25280 P14151 leukocytesurface leu-8 antigen; GP90-MEL; leukocyte-endothelial cell adhesionmolecule 1 (LECAM1); CD62L antigen; SELL LUCA2; lysosomal hyaluronidase2 (HYAL2); PH-20 homolog U09577 Q12891 lymphocyte antigen M81141 Q30099lymphoid-restricted homolog of SP100 protein (LYSP100) U36500 Q13342lymphotoxin-beta (LT-beta; LTB); tumor necrosis factor C (TNFC) L11015Q06643 lysosomal acid lipase/cholesteryl ester hydrolase precursor(LAL); acid cholesteryl ester hydrolase; M74775 P38571 sterol esterase;lipase A (LIPA); cholesteryl esterase lysosomal pro-X carboxypeptidaseL13977 P42785 macrophage colony stimulating factor I receptor precursor(CSF-1-R); fms proto-oncogene (c-fms); X03663 P07333 CD115 macrosialinprecursor S57235 P34810 manic fringe U94352 O00587 matrixmetalloproteinase 17 (MMP17); membrane-type matrix metalloproteinase 4(MT-MMP4) X89576 Q14850 matrix metalloproteinase 9 (MMP9); gelatinase B;92-kDa type IV collagenase precursor (CLG4B) J05070; D10051 P14780 MHCclass II HLA-DR-beta (DR2-DQW1/DR4 DQW3) precursor M20430 Q30166microsomal aminopeptidase N; myeloid plasma membrane glycoprotein CD13M22324 P15144 microtubule-associated protein 1B L06237 P46821 migrationinhibitory factor-related protein 14 (MRP14); calgranulin B; leukocyteL1 complex heavy X06233 P06702 subunit; S100 calcium-binding protein A9migration inhibitory factor-related protein 8 (MRP8); calgranulin A;leukocyte L1 complex light subunit; X06234 P05109 S100 calcium-bindingprotein A8; cystic fibrosis antigen (CFAG) myeloid cell nucleardifferentiation antigen (MNDA) M81750 P41218 myotonin-protein kinase;myotonic distrophy protein kinase (MDPK); DM-kinase (DMK) L19268 Q09013neurogenic locus notch protein (N) M99437 Q04721 neurogranin (NRGN); RC3Y09689 Q92686 neurotrophic tyrosine kinase receptor-related 3; TKTprecursor X74764 Q16832 neutrophil cytosol factor 2; neutrophil NADPHoxidase factor 1 (NCF1); p47-PHOX); 47-kDa autosomal M25665 P14598chronic granulomatous disease protein neutrophil gelatinase-associatedlipocalin precursor (NGAL); 25-kDa alpha-2-microglobulin-related X99133P80188 subunit of MMP9); lipocalin 2; oncogene 24P3 ninjurin-1 U72661Q92982 NKG5 protein precursor; lymphokine LAG2; T-cell activationprotein 519 X54101 P22749 NT-3 growth factor receptor precursor (NTRK3);C-trk tyrosine kinase (TRKC) U05012 Q16288; Q16289; Q12827 nuclearreceptor-related 1 X75918 P43354 NuMA Z11583 Q14981 osteoclaststimulating factor U63717 Q92882 P126 (ST5) U15131 P78524 P2Xpurinoceptor 1; ATP receptor P2X1 X83688 P51575 P2X purinoceptor 5(P2X5) AF016709 Q93086 paxillin U14588 P49023 PC8 precursor U33849Q16549 peripheral myelin protein 22 (PMP22); CD25 protein; SR13 myelinprotein D11428 Q01453 peroxisomal bifunctonal enzyme L07077 Q08426phenol-sulfating phenol sulfotransferase 1 (PPST1); thermostable phenolsulfotransferase (TS-PST); U09031 + U28170 + L19956 P50225 +HAST1/HAST2; ST1A3; STP1 + PPST2; ST1A2; STP2 + monoamine-sulfatingphenol sulfotransferase P50226 + P50224 phospholipase C beta 2 (PLC-beta2; PLCB2); 1-phosphatidylinositol 4,5-bisphosphate M95678 Q00722phosphodiesterase beta 2 phosphoribosyl pyrophosphate synthetase subunitI D00860 P09329 PIG7 AF010312 Q99732 pim-1 proto-oncogene M54915 P11309platelet basic protein precursor (PBP); connective tissue activatingpeptide III (CTAP III); low-affinity M54995; M38441 P02775 plateletfactor IV (LA PF4); beta thromboglobulin (beta TG); neutrophilactivating peptide 2 (NAP2) platelet endothelial cell adhesion moleculeHS78146 P16284 platelet membrane glycoprotein IIB precursor (GP2B);integrin alpha 2B (ITGA2B); CD41 antigen M34480; J02764 P08514 plateletmembrane glycoprotein IIIA precursor (GP3A); integrin beta 3 (ITGB3);CD61 antigen J02703; M25108 P05106; Q13413; Q16499 platelet-activatingfactor receptor (PAFR) D10202 P25105 platelet-derived growth factor Asubunit precursor (PDGFA; PDGF-1) X06374 P04085 PRB-binding proteinE2F1; retinoblastoma-binding protein 3 (RBBP3);retinoblastoma-associated M96577 Q01094; protein 1 (RBAP1); PBR3 Q92768;Q13143 prostaglandin G/H synthase 1 P23219 protein-tyrosine phosphatase1C (PTP1C); hematopoietic cell protein-tyrosine phosphatase; SH-PTP1X62055 P29350 prothrombin precursor; coagulation factor II V00595 P00734proto-oncogene tyrosine-protein kinase Ick; p56-Ick; lymphocyte-specificprotein tyrosine kinase (LSK); U07236 P06239 T-cell-specificprotein-tyrosine kinase P-selectin precursor (SELP); granule membraneprotein 140 (GMP140); PADGEM; CD62P antigen; M25322 P16109leukocyte-endothelial cell adhesion molecule 3 (LECAM3) purine-richsingle-stranded DNA-binding protein alpha (PURA) M96684 Q00577 rabgeranylgeranyl transferase alpha subunit Y08200 Q92696 rabgeranylgeranyl transferase beta subunit Y08201 P53611; Q92697 RaIBGTP-binding protein M35416 P11234 ras-related C3 botulinum toxinsubstrate 2; p21-rac2; small G protein M64595; M29871 P15153 ras-relatedprotein RAB5A M28215 P20339 related to receptor tyrosine kinase (RYK)S59184 P34925 replication protein A 70-kDa subunit (RPA70; REPA1; RF-A);single-stranded DNA-binding protein M63488 P27694 rho GDP dissociationinihibitor 2 (RHO GDI2; RHO-GDI beta); LY-GDI; ARHGDIB; GDID4 L20688P52566 rho-GAP hematopoietic protein C1 (RGC1); KIAA0131 X78817 P98171rho-related GTP-binding protein (RHOG); ARHG X61587 P35238 ribonuclease6 precursor U85625 O00584 ribosomal protein S6 kinase II alpha 1(S6KII-alpha 1); ribosomal S6 kinase 1 (RSK1) L07597 Q15418 S100calcium-binding protein A1; S-100 protein alpha chain X58079 P23297SCGF-beta D86586 BAA21499 SEC7 homolog B2-1 M85169 Q15438 selectin Pligand U02297 Q14242; Q12775 semaphorin; CD100 U60800 Q92854 serumresponse factor (SRF) J03161 P11831 SH3-binding protein 2 AF000936P78314 signaling inositol polyphosphate 5 phosphatase; SIP-110 U50040Q13544 sonic hedgehog (SHH) L38518 Q15465 specific 116-kDa vacuolarproton pump subunit U45285 Q13488 steroid 5-alpha reductase 1 (SRD5A1);3-oxo-5-alpha steroid 4 dehydrogenase 1 M32313; M68886 P18405 stromalcell derived factor 1 receptor (SDF1 receptor); fusin; CXCR4;leukocyte-derived seven D10924 P30991 transmembrane domain receptor(LESTR); LCR1 superoxide dismutase 2 M36693 P04179 T-cell surfaceglycoprotein CD3 epsilon subunit precursor; T-cell surface antigenT3/leu-4 epsilon X03884 P07766 subunit (T3E) tenascin precursor (TN);hexabrachion (HXB); cytotactin; neuronectin; GMEM; miotendinous antigen;X78565; M55618 P24821; glioma-associated extracellular matrix antigenQ15567; Q14583 thrombospondin 1 precursor (THBS1; TSP1) X14787 P07996thymidine phosphorylase precursor (TDRPase); platelet- derivedendothelial cell growth factor M63193 P19971; (PDECGF); gliostatinQ13390 TNF-related apoptosis inducing ligand (TRAIL); APO-2 ligand(APO2L) U57059 P50591 TRAIL receptor 3; decoy receptor 1 (DCR1) AF016267O14755 transcription factor Spi-B X66079 Q01892 transcriptionalregulator interferon-stimulated gene factor 3 gamma subunit (ISGF3G);interferon-alpha M87503 Q00978 (IFN-alpha) responsive transcriptionfactor subunit transforming growth factor-beta 3 (TGF-beta3) J03241P10600 tuberin; tuberous sclerosis 2 protein (TSC2) X75621 P49815 type Icytoskeletal 18 keratin; cytokeratin 18 (K18) M26326 P05783 type IIcytoskeletal 6 keratin: cytokeratin 6A (CK6A); K6A keratin (KRT6A) +CK6B; KRT6B + CK6C; J00269 + L42592 + L42601 + P02538 KRT6C + CK6D;KRT6D + CK6E; KRT6E + CK6F; KRT6F L42610 + L42611 + L42612tyrosine-protein kinase lyn M16038 P07948 tyrosine-protein kinasereceptor UFO precursor; axl oncogene M76125 P30530 vascular endothelialgrowth factor B precursor (VEGFB) + VEGF-related factor isoform VRF186U48801; U43369 P49765 vav oncogene X16316 P15498 v-erbA related protein(EAR2) X12794 P10588 versican core protein precursor; large fibroblastproteoglycan; chondroitin sulfate proteoglycan core U16306; X15998;P13611 protein 2; glial hyaluronate-binding protein (GHAP) U26555;D32039 vitamin K-dependent protein S Y00692 P07225

For example, it was found that 17 of the genes differentially expressedin human neointima encode transcriptional regulators. mRNA levels for 14transcription factors were induced in neointima and 3 showed a decreasedexpression (FIG. 15). Some transcription factors of the former grouphave previously been related to proliferation and apoptosis of SMCs,such as HMG-1, E2F1, IRF-1, Fli-1, and with pro-inflammatory signalingin human neointima, such as IRF-1, IRF-7 and RelB. The followingtranscription factors were upregulated: E2F1, estrogen-related receptoralpha, ets domain protein elk-3, fli-1 oncogene, HMG-1, interferonregulatory factor 1, interferon regulatory factor 7, ISGF3-gamma,nuclear receptor-related 1, RELB, transcription factor Spi-B, vavoncogene, v-erbA related protein, vitamin D3 receptor; whereas thefollowing were downregulated: homeobox protein HOXB7, early growthresponse protein 1, serum response factor.

Striking changes seem to take place in the expression of transcriptionfactors of the Ets family. Whereas Spi-B, the fli-oncogene, and theEts-repressor Elk-3 were induced in neointima, the Ets transcriptionfactor Egr-1 was repressed (FIGS. 14 and 15).

Furthermore, a number of genes involved in controlling or mediatingproliferative responses were differentially expressed between neointimaand control groups. The platelet-derived growth factor (PDGF)-A andangiotensinogen genes, whose products act on SMCs as mitogens, wereexclusively expressed in neointima (FIG. 14). Angiotensin is known to beupregulated by insulin and to induce the expression of PDGF-A in SMCs.As a sign of ongoing proliferation, several genes known to be expressedwith the G1/S transition of the cell cycle were found to be upregulatedin neointima. Those include transcription factor E2F1, 70-kDareplication protein A, oncogene product Pim-1 and geranylgeranyltransferase. In addition, upregulation of the cell-cycle regulatedhistone H4, which is expressed in the G/S1 and S-phase of the cell cycleindicating ongoing proliferation in human neointima, was observed.

Reprogramming of cell growth in neointima evidently led to induction ofseveral genes in neointima encoding proteins with functions in differentsignal transduction pathways, including the cell surface receptorsEDG-1, EDG-4, insulin receptor and P2X purinoceptor 5, and othersignaling proteins like the ribosomal protein S6 kinase II alpha 1,farnesyltransferase, phospholipase C beta 2, growth factorreceptor-bound protein 2, and the small G proteins CDC42, RhoG, p21-Rac2and RalB. The enzyme farnesyltransferase catalyzes the essentialpost-translational lipidation of Ras and several other signaltransducing G proteins. G proteins, like p21-Rac2, CDC42 and RhoG playpivotal roles in signal transduction pathways leading to cell migrationand cell proliferation. Likewise, agonist-stimulated 1,4,5-triphosphate(IP3) production by phospholipase C beta 2 in smooth muscle requires Gprotein activation and activated Rac and Cdc42 associate with PI 3kinase that plays an important role in the activation of the p70 S6kinase. The p70 S6 kinase (p70S6K) is an important regulator of cellcycle progression to enter G1 phase and to proceed to S phase inresponse to growth factors and mitogens. It is involved in multiplegrowth factor related signal transduction pathways that are known toplay pivotal roles in neointima formation, like angiotensin, endothelinand PDGF. In line with upregulation of p70 S6 kinase, significantupregulation of the FK506-binding protein (FKBP) 12 at mRNA (FIG. 14)and protein level in neointima was found.

It was observed that a number of genes encoding inhibitors of cell cycleprogression were expressed in quiescent media but significantlydownregulated in neointima (FIG. 14). Those included CIP1, p16-INK4,metallothionein, TGF-beta3, mammary-derived growth inhibitor, FrzB andthe Gadd45 beta and gamma subunits.

Additionally, upregulation of genes in human neointima encoding proteinswith pro-apoptotic function, like caspase-1, DAP-1 and APO-2 ligand, aswell as upregulation of genes encoding proteins with anti-apoptoticfunction, like BAG-1, BCL-2-related protein A1 and the Trail receptor 3(FIG. 14) was found.

Finally, the human neointima transcriptome showed upregulation of 32genes related to IFN-γ signaling (FIG. 16). The IFN-γ receptor alpha wasexpressed in neointima, proliferating CASMCs and—to a lesser degree—inblood cells; whereas the IFN-γ receptor beta was mainly expressed inneointima specimen. Likewise, an upregulation of Pyk2 was observed.

Upregulation of the IFN-γ regulated genes for caspase-1, caspase-8 andDAP-1 was found in human neointima. However, mRNAs for theanti-apoptotic proteins BAG-1, Pim-1 (both regulated by IFN-γ) andBCL-2-related protein A1 were also upregulated in neointima versuscontrol (FIG. 14).

Numerous genes with functions in inflammatory responses were foundactivated in human neointima. Pro-inflammatory gene patterns came frominfiltrating inflammatory cells such as macrophages and T lymphocytes(e.g., CD11b, CD3) (FIG. 14C) or from neointimal SMCs (e.g.,prostaglandin G/H synthase 1, phospholipase A2, heat shock protein 70,C5a anaphylatoxin receptor, IFN-γ receptor) (FIG. 14A and B).

The selective expression of CD40 in neointima deserves attention (FIG.14A). CD40 is a member of the TNF receptor family that was initiallydescribed on the surface of B cells.

The following cytoskeletal, extracellular matrix and cell adhesionchanges in neointima were observed:

An upregulation of connexin43 and of cytokeratin-18 in neointima as isseen in proliferating CASMC (FIG. 14B, upper panel), whereas theexpression of desmin was strongly reduced in neointima (FIG. 14D, upperpanel).

Whereas the transcription of different collagen subtypes and tenascinwere reduced in neointima (FIG. 14D, upper panel), expression ofthrombospondin-1 and versican were upregulated (FIG. 14B, upper panel).

A number of genes encoding adhesion molecules, including P-selectin,ICAM2 and cadherin 16, were found highly expressed in neointima but notin SMCs, blood cells or control vessels (FIG. 14A, upper panel). Anumber of other adhesion molecules were similarly expressed inneointima, cultured SMCs (FIG. 14B) and blood cells (FIG. 14C).Neointima appears to downregulate expression of certain adhesionmolecules that are normally expressed in media/intima of arteries, suchas integrins α7B, α3 or MUC18.

EXAMPLE VII Upregulated Genes of the IFN-γ Signaling Pathway

As shown herein above, the expression of 2,435 genes of known functionin atherectomy specimen of 10 patients with in-stent restenosis, bloodcells of 10 patients, normal coronary artery specimen of 11 donors, andcultured human coronary artery smooth muscle cells was investigated and224 genes that were differentially expressed with high statisticalsignificance (p<0.03) between neointima and control tissue wereidentified. In particular, 32 upregulated genes that are related tointerferon-γ signaling were identified in neointima.

The IFN-γ receptor alpha was expressed in neointima, proliferatingCASMCs and—to a lesser degree—in blood cells; whereas the IFN-γ receptorbeta was mainly expressed in neointima specimen.

IFN-γ signals via a high-affinity receptor containing an α- andβ-receptor chain. Interstingly, TH1 cells use receptor modification toachieve an IFN-γ-resistant state (Pernis, Science 269 (1995), 245–247).The subtype-specific difference in the activation of the IFN-γ signalingpathway of type 1 and type 2 T helper cells is due to a lack of IFN-γreceptor β in type 1 T cells. Therefore, the here presented data wouldargue that a high affinity IFN-γ-receptor containing both chains ismainly expressed in smooth muscle cells of the neointima.

Consistent with an activation of IFN-γ signaling, upregulation of twotranscription factors in neointima that are essential for IFN signallingwere found: IRF-1 and ISGF3γ (p48). These transcription factors areknown to be transcriptionally upregulated by IFN-γ (Der, Proc. Natl.Acad. Sci. 95 (1998), 15623–15628), and both are key players in IFN-γsignalling (Matsumoto, Biol. Chem. 380 (1999), 699–703; Kimuar, GenesCells 1 (1996), 115–124; Kirchhoff, Nucleic Acids Res. 21 (1993),2881–2889; Kano, Biochem. Biophys. Res. Commun. 257 (1999), 672–677).Likewise, upregulation of the tyrosine kinase Pyk2 was observed, whichhas been shown to play a role in the signal transduction by angiotensinin SMCs (Sabri, Circ. Res. 83 (1998), 841–851). Pyk2 is selectivelyactivated by IFN-γ and inhibition of Pyk2 in NIH 3T3 cells results in astrong inhibition of the IFN-γ-induced activation of MAPK and STAT1(Takaoka, EMBO J. 18 (1999), 2480–2488.

A key event in IFN-γ-induced growth inhibition and apoptosis is theinduction of caspases (Dai, Blood 93 (1999), 3309–3316). It has beenshown that IRF-1 induces expression of caspase-1 leading to apoptosis invascular SMCs (Horiuchi, Hypertension 33 (1999), 162–166), and thatapoptotic SMCs and macrophages colocalize with caspase-1 inatherosclerosis (Geng, Am. J. Pathol. 147 (1995), 251–266). In thisstudies, upregulation of the IFN-γ-regulated genes for caspase-1,caspase-8 and DAP-1 in human neointima was found. However, mRNAs for thethe anti-apoptotic proteins BAG-1, Pim-1 (both regulated by IFN-γ) andBCL-2-related protein A1 were also upregulated in neointima versuscontrol (FIG. 16), supporting the notion that proliferation andapoptosis occur simultaneously in human neointima with a preponderanceof proliferation.

Coordinated regulation of genes whose products act at different steps inthe neointima process was a recurring theme of our gene expressionanalysis. Regarding the IFN-γ pathway, not only the genes for thecomplete receptor, the main transcription factors, components of thesignal transduction pathway (Dap-1, BAG-1, Pim-1, IFN-γ-inducibleprotein, IFN-inducible protein 9–27) were induced but also severaltarget genes of the IFN-γ pathway, like CD40, CD13 and thrombospondin-1(FIG. 16).

The IFN-γ-regulated gene cluster was expressed in the neointima specimenbut some of the relevant genes, like IRF-1, were also expressed in bloodsamples. To identify the cell type that predominantly contributed to theIFN-γ regulated pattern, frozen sections of neointima specimen fromcoronary in-stent restenosis (n=3) and from restenosis of peripheralarteries (n=6) were stained with antibodies specific for IRF-1. Thisprotein was chosen because it is an essential component of the IFN-γsignal transduction pathway (Kimura, loc. cit.) and was expressedcoordinately with the other genes in the cluster (FIG. 16).Immunohistochemical analysis showed strong nuclear and cytoplasmicstaining of IRF-1 in neointimal SMCs of a carotid restenosis (FIG. 17)and of coronary in-stent restenosis (FIG. 18), as identified by theirspindle-shaped nuclei and by staining with the smooth muscle cell markeralpha-actin (FIG. 18). The nuclear staining of IRF-1 in in-stentrestenosis (FIG. 18) indicated that the IRF-1 transcription factor isalso activated. SMCs in control media of carotid arteries did not showIRF-1 staining (FIG. 17). CD3-positive cells were much less abundant inthe specimen (FIG. 18) than SMCs (FIG. 18), indicating that SMCscontributed mostly to the increased IRF-1 expression in human neointima.

The presence of IFN-γ in human atherosclerotic lesions is wellestablished (Ross, N. Engl. J. Med. 340 (1999), 115–126) although itsrole remains unclear. Whereas IFN-γ inhibits proliferation and inducesapoptosis in SMCs in vitro (Horiuchi, loc. cit.; Warner, J. Clin. Invest83 (1989), 1174–1182), absence of IFN-γ reduces intima hyperplasia inmouse models of atheroma and transplant arteriosclerosis (Gupta, J.Clin. Invest 99 (1997), 2752–2761; Raisanen-Sokolowski, Am. J. Pathol.152 (1998), 359–365). In line with this observation, it was shown thatIFN-γ induces arteriosclerosis in absence of leukocytes in pig and humanartery tissues by their insertion into the aorta of immunodeficient mice(Tellides, Nature 403 (2000), 207–211).

The role of infiltrating T lymphocytes in neointima of in-stentrestenosis has not been examined yet. In this study it was shown thatCD3-positive cells can be detected by immunobiochemists in 3 out of 4neointima samples (see FIG. 18), and a CD3-specific hybridization signalon cDNA arrays with 7 out of 10 neointima specimen was obtained (FIG.18). IFN-γ-related expression patterns were also observed in samplesnegative for CD3 as examined by either method, suggesting that thecytokine could act on neointima in a paracrine fashion over somedistance with no need for massive T cell infiltration. While T cells andthe pro-inflammatory cytokine IFN-γ are known to play an important rolein atherosclerosis (Ross, loc. cit.), their role in the development ofneointima is largely unexplored. The here provided data suggest animportant role of IFN-γ in the pathophysiology of neointimalhyperplasia.

EXAMPLE VIII Preparation of a Surrogate Cell Line

A surrogate cell line for a pathologically modified cell and/or tissuemay be prepared by the following steps:

a) Definition of the Transcriptome/Gene Expression Pattern of theDiseased Tissue:

-   -   Microscopic specimen of diseased tissue may be obtained by        either atherectomy, debulking, biopsy, laser dissection of        diseased tissue or macroscopic surgical dissection of diseased        tissue. After acquisition, microscopic specimen are immediately        frozen in liquid nitrogen and kept in liquid nitrogen until mRNA        preparation is performed in order to preserve the in vivo status        of the samples' transcriptomes.    -   The cells in such samples express a particular set of genes        which is reflected by the presence of distinct mRNA molecules        occuring at various concentrations. The entirety of mRNA        molecules and their relative amounts in a given clinical sample        is referred to as the transcriptome. The transcriptome of a        diseased tissue is expected to be different from that of a        healthy tissue. The differences relate to the up- or        downregulated expression of genes involved in causing,        maintaining or indicating the diseased state of the tissue. The        analysis of the transcriptome is typically limited by the number        of cDNA elements a particular array carries.    -   mRNA preparation and amplification is carried out according to        the method of the invention and described herein above.    -   In particular, microscopic specimen of diseased tissue are        quick-frozen and kept in liquid nitrogen until mRNA preparation        and cDNA synthesis is performed as described herein above.        Frozen tissue is ground in liquid nitrogen and the frozen powder        dissolved in Lysis buffer according to the procedure of RNA        preparation. The lysate is centrifuged for 5 min at 10,000 g at        4° to remove cell debris. RNA can be prepared as total RNA or as        mRNA as described ein (Schena, Science 270 (1995), 467–470), in        Current Protocols, in the Clontech manual for the Atlas cDNA        Expression Arrays or as described in (Spirin, Invest. Ophtalmol.        Vis. Sci. 40 (1999), 3108–3115), as described in (Chee, Science        274 (1996), 610–614; Alon, Proc. Natl. Acad. Sci. 96 (1999),        6745–6750; Fidanza, Nucleosides Nucleotides 18 (1999),        1293–1295; Mahadevappa, Nat. Biotechnol. 17 (1999), 1134–1136;        Lipshutz, Nat. Genet. 21 (1999), 20–24) for the Affymetrix        arrays or as described by Qiagen.

cDNA preparation and labeling can be performed-as described by Clontechor Affymetrix in the user's manual for the arrays hybridization kits oras described in (Spirin, loc. cit.; Chee, loc. cit.; Alon, loc. cit.;Fidanza, loc. cit.; Mahadevappa, loc. cit.; Lipshutz, loc. cit.).Additionally, amplified cDNA can be used. Preparation of cDNAamplificates and labeling of amplificated cDNA can be performed asdescribed herein above or by Spirin (loc. cit.).

-   -   Obtained, labeled cDNA can be employed in hybridization assays.        Hybridization of labeled cDNA and data analysis can be performed        under conditions as described in the user's manual from        Clontech's Atlas™ cDNA Expression Arrays User Manual or in the        manufacter's manual of Affymetrix or as described by (Spirin,        loc. cit.; Chee, loc. cit.; Alon, loc. cit.; Fidanza, loc. cit.;        Mahadevappa, loc. cit.; Lipshutz, loc. cit.).        b) Definition of the Transcriptome/Gene Expression Pattern of        Control Tissue    -   To identify disease-specific gene expression patterns, the gene        expression pattern of the diseased tissue can be compared to        control material from healthy donors. In the case of atherectomy        material this can be healthy media and intima of non-elastic,        i.e., muscular arteries. In the case of heart muscle biopsies or        kidney biopsies, healthy control tissue can be used that is        collected in the course of the operation. Additionally, gene        expression pattern of cells of neighbouring unaffected tissue or        of infiltrating cells, like blood, cells can be analyzed. Based        upon the celluar characterization of a tissue by        immunohistochemical analysis using antibodies to cell marker        proteins, transcriptome can be determined from cultured human        cell lines of the same type. (Example: arteries stain positive        for smooth muscle cells and endothelial cells; consequently        transcriptomes are obtained from cultured human smooth muscle        and endothelial cells).    -   mRNA preparation and amplification can be carried out as        described herein above and in accordance with the method of the        present invention. Obtained (labeled) cDNA may be employed in        hybridization assays as described herein above.        c) Determination of a Relevant Set of Disease Specific Genes    -   To determine disease-specific gene expression patterns first the        gene expression pattern of the diseased tissue should be        compared to the gene expression pattern of healthy control        tissue. For comparison, the mean expression value of at a        sufficient number of diseased specimen (e.g., 10) and the same        number of control specimen should be compared. Genes with an        expression ratio >2.5-fold between the the two groups should be        analyzed for their relative expression in one group: there        should be >5/10 positive in one group, if there are 0/10 in the        other or at least 7/10 in one group if there are maximally 3/10        positive in the other group. Additionally, these data should be        analysed statistically to define genes with an p<0.05 with e.g.        the Wilcoxon test as described in the manual of SPSS 8.0.    -   Genes selected based upon their significant over- or        underexpression by a factor of 2.5 are refered to as aberrantly        regulated in the diseased tissue, or as diseases-related genes.        Disease-related genes genes are then grouped by the functions of        encoded proteins. e.g. genes encoding proteins of the signalling        pathway, cytokines, chemokines, hormones, their receptors,        proteins specific or infiltrating cells, or proteins involved in        extracellular matrix, cell adhesion, migration, cell division,        cell cycle arrest. Likewise genes of unknown function, as        available thorugh public EST data bases, can be identified as        being disease-related.        d) Screen for a Cell Line with a Transcriptome Most Closely        Resembling that of Diseased Tissue    -   Drugs that can potentially regulate the expression of diseased        genes can be discovered by screening large libraries of        chemicals or biologics. In order to identify such drugs, a        screening cell line must be available that faithfully reflects        the transcriptome of the diseased tissue and is avaiabale in        large quantaties for the performance of a comprehensive drug        screen. Moreover information is needed of how the drug candidate        should alter the transcriptome of the cell line that has        characteristics of the transcriptome of the diseased tissue.        This information is obtained from the transcriptome of the        healthy control tissue. The drug should be able to re-estsblish        features of a “healthy” transcriptome.    -   A human cell line, which is most similiar to the cellular origin        of the diseased tissue, e.g, coronary artery smooth muscle cells        for atherectomy, HepG2 cells for liver diseases, renal cells for        kidney diseases or cardiomyoblasts for heart muscle disease        should be used. Cells should be grown under standard conditions        as described in the manufacter's manual like the ones from ATCC.    -   Transcriptome analysis/gene expression pattern analysis can be        performed as described for the diseased and the control tissue        and gene expression pattern should be compared to the gene        expression pattern of the diseased and the healthy tissue. For        generating a surrogate screening cell line, the cell line which        shows a transcriptome most similar to the diseased transcriptome        should be selected.        e) Adaptation of a Cell Line to Mimick Diseased        Transcriptome/Gene Expression Pattern    -   In order to generate a surrogate screening cell line for the        diseased tissue, it may be necessary to adapt the transcriptome        of the selected cell line to the transcriptome of the diseased        tissue. This can on the one hand be achieved by incubation of        the cell line with compounds such as cytokines or hormones, that        had been shown to play an important role in the gene expression        pattern of the diseased tissue. Likewise such compounds can be        identified by transcriptome analysis of diseased tissue as        exemplified with neoinitima where evidence for a role of        interferon-gamma was obtained. Instead of addition of compounds        with relevance for the disease, the screening cell line can be        conditioned by co-culture with other cell types relevant for the        pathophysiology of the disease. Such cells can for instance be        inflammatory cells, like macrophages or T cells, that migrate        into the diseased tissue and by released factors or        cell-cellcontact contribute to the disease-specific gene        expression pattern. In each case, transcriptome analysis of the        surrogate line must identify the optimal addition to generate a        disease-specific expression pattern.    -   Compounds that can be used for adapting the transcriptome of a        surogate cell line to the diseased state comprise cytokines,        growth factors, small molecule compounds (drugs), or peptides        and peptidomimetics. Cell lines that can be used for such an        adaptation comprise human monocytic cell lines, like U937, THP-1        or Monomac-6, or human T-cell lines like Jurkat.    -   The co-culture/treatment conditions leading in the surrogate        cell line to a state closest to the diseased transcriptome are        selected for drug screening.

In the following, a specific example should illustrate the preparationof a surrogate. In particular, a surrogate cell (line) for restenotictissue is prepared by the following steps:

a) Aquisition of In-stent Restenotic Tissue

Patients

-   -   The in-stent restenosis study group consisted of 13 patients who        underwent separate atherectomy procedures by X-sizer within the        renarrowed stent between 4–23 month after primary stent        implantation. All patients gave informed consent to the        procedure and received 15,000 units heparin before the        intervention followed by intravenous heparin infusion, 1,000        units/h for the first 12 h after sheat removal as standard        therapy. All patients received aspirin, 500 mg intravenously,        before catherisation, and postinterventional antithrombotic        therapy consisted of Aticlopidine (250 mg bds) and aspirin (100        mg bds) throughout the study.

Sample Preparation

-   -   Atherectomy specimen were immediately frozen in liquid nitrogen        after debulking of the lesion, and kept in liquid nitrogen until        mRNA preparation was performed as described. For histology and        immunhistochemistry of the in-stent restenotic tissue from        coronary arteries (n=3), the samples were fixed in 4%        paraformaldehyd and embedded in paraffin as described.

Morphological Characterization of Restenotic Tissue

-   -   Immunohistochemistry for cell typing was performed on        paraffin-embedded sections of three neointima specimen from        coronary in-stent restenosis and, for detection of FKBP12, on        frozen sections of four neointima specimen from carotid        restenosis. Three μm serial sections were mounted onto DAKO        ChemMate™ Capillary Gap Microscope slides (100 μm) baked at        65° C. overnight, deparaffinized and dehydrated according to        standard protocols. For antigen retrieval, specimens were boiled        4 min in a pressure cooker in citrate buffer (10 mM, pH 6.0).        Endogenous peroxidase was blocked by 1% H2O2/methanol for 15        minutes. Unspecific binding of the primary antibody was reduced        by preincubation of the slides with 4% dried skim milk in        Antibody Diluent (DAKO, Denmark). Immunostaining was performed        by the streptavidin-peroxidase technique using the ChemMate        Detection Kit HRP/Red Rabbit/Mouse (DAKO, Denmark) according to        the manufacturer's description. The procedures were carried out        in a DAKO TechMate™ 500 Plus automated staining system. Primary        antibodies against smooth muscle actin (M0635, DAKO, Denmark;        1:300), CD3 (A0452, DAKO, Denmark; 1:80), MAC387 (E026, Camon,        Germany; 1:20) and FKBP12 (SA-218, Biomol, Germany, 1:20) were        diluted in Antibody Diluent and incubated for 1 h at room        temperature. After nuclear counterstaining with hematoxylin, the        slides were dehydrated and coverslipped with Pertex (Medite,        Germany).

The Cellular Composition of Debulked In-stent Restenotic Material

-   -   Representative samples obtained from x-sizer treatment of a        neointimal hyperplasia were analyzed by immunhistochemistry in        order to determine its cellular composition. FIG. 7A shows an        E.-van-Giesson staining of a section cut from a small sample of        debulked restenotic material. With this staining procedure,        collagen fibers stain red, fibrin stains yellow and cytoplasm of        smooth muscle cells stains dark-yellow-brown. The majority of        the volume of debulked material was composed of loose        extracellular matrix-like collagen fibers stained in light red.        Yellow fibrin staining was barely detectable. Cells with        spindle-shaped nuclei and a yellow/brown-stained cytoplasm were        frequent. Their identity as smooth muscle cells and their high        abundance in restenotic material was supported by immunostaining        with an antibody against smooth muscle α-actin (FIG. 7B). There,        the staining pattern of a section from an entire specimen as        used for gene expression analysis is shown. As described below,        such samples also gave raise to a strong smooth muscle-specific        α-actin mRNA signal (see FIG. 8). These results support findings        from previous studies (Kearney, Circulation 95 (1997),        1998–2002; Komatsu, Circulation 98 (1998), 224–233; Strauss, J.        Am. Coll. Cardiol. 20 (1992), 1465–1473) demonstrating that the        main cell type found in neointima is derived from smooth muscle        cells. As described in the literature (Kearney, loc. cit.;        Komatsu, loc. cit.; Strauss, loc. cit.) mononuclear infiltrates        could also be identified in some areas of debulked restenotic        tissue specimen. These infiltrates consisted mainly of        macrophages and to a lesser degree of t-lymphocytes. No        b-lymphocytes were detectable in the restenotic tissue by using        an antibody against CD20 for immunhistochemical staining.        b) Transcriptome Analysis of Restenotic Material    -   Transcriptome analysis of neointima was performed using the        method of mRNA amplification as described herein above.

mRNA Preparation

-   -   Microscopic specimen diseased tissue were quick-frozen and kept        in liquid nitrogen until. mRNA preparation and cDNA synthesis        was performed. Frozen tissue is ground in liquid nitrogen and        the frozen powder dissolved in Lysis/Binding buffer (100 mM        Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, pH 8.0, 1% LiDS, 5 mM        dithiothreitol (DTT)) and homogenized until complete lysis is        obtained. The lysate is centrifuged for 5 min at 10,000 g at 4°        to remove cell debris. mRNA is prepared using the Dynbeads® mRNA        Direct Kit™ (Dynal, Germany) following the manufacture's        recommendation. Briefly, lysate was added to 50 μL of pre-washed        Dynabeads Oligo (dT)25 per sample and mRNA was annealed by        rotating on a mixer for 30 min at 4° C. Supernatant was removed        and Dynabeads Oligo (dT)25/mRNA complex was washed twice with        washing buffer containing Igepal (50 mM Tris-HCl, pH 8.0, 75 mM        KCl, 10 mM DTT, 025% Igepal), and once with washing buffer        containing Tween-20 (50 mM Tris-HCl, pH 8.0, 75 mM KCl, 10 mM        DTT, 0.5% Tween-20).

Preparation of Amplified cDNA

cDNA is amplified by PCR using the procedure of Klein et al. (C. Kleinet al.). First-strand cDNA synthesis is performed as solid-phase cDNAsynthesis. Random priming with hexanucleotide primers is used forreverse transcription reaction. mRNAs are each reversely transcribed ina 20 μL reaction volume containing 1× First Strand Buffer (Gibco), 0.01M DTT (Gibco), 0.25% Igepal, 50 μM CFL5c-Primer (SEQ ID NO:8) [5′-(CCC)5GTC TAG A (NNN)2-3′], 0.5 mM dNTPs each (MBI Fermentas) and 200 USuperscript II (Gibco), and incubate at 44° C. for 45 mm. A subsequenttailing reaction is performed in a reaction volume of 10 μL containing 4mM MgCl2, 0.1 mM DTT, 0.2 mM dGTP, 10 mM KH2PO4 and 10 U of terminaldeoxynucleotide transferase (MBI Fermentas). The mixture is incubatedfor 24 mm at 37° C.

cDNA is amplified by PCR in a reaction volume of 50 μL containing 1×buffer 1 (Expand™ Long Template PCR Kit, Boehringer Mannheim), 3%deionized formamide, 120 μM CP2-Primer (SEQ ID NO:14) [5′-TCA GAA TTCATG (CCC)5-3′], 350 μM dNTP and 4.5 U DNA-Polymerase-Mix (Expand™ LongTemplate PCR Kit, Roche Diagnostics, Manuhein). PCR reaction isperformed for 20 cycles with the following cycle parameters: 94° C. for15 sec, 65° C. for 0:30 min, 68° C. for 2 min; for another 20 cycleswith: 94° C. for 15 sec, 65° C. for 30 sec, 68° C. for 2:30+0:10/cyclemin; 68° C. 7 min; 4° C. forever.

Expression of Specific Genes in Microscopic Human Tissue Samples

-   -   In order to optimally preserve the in situ mRNA levels,        restenotic and control specimen were immediately frozen after        harvest in liquid nitrogen and carefully lyzed as described in        Materials and Methods. After PCR amplification of the        synthesized cDNA the amount of the amplified cDNA was measured        by a dot blot assay and found to be between 200–300 ng/μl. The        quality of every amplified cDNA sample was tested by        gene-specific PCR using primers detecting cDNAs for β-actin,        smooth muscle cell α-actin and the ubiquitous elongation factor        EF-1α. FIG. 8 shows a representative result with material from        patient B and control media from donor b. In both specimen, PCR        signals of the correct size from house-keeping genes β-actin and        EF-1α were detectable in equivalent amounts (compare lanes 1 and        2 with lanes 4 and 5). Additionally, α-actin signals as marker        for smooth muscle cells was obatined from each specimen (lanes 3        and 6). These results show that mRNA prepraration, cDNA        synthesis and PCR amplification of cDNA is feasible with        microscopic human restenosis samples.

Dig-dUTP Lab ling of cDNA Probes

-   -   25 ng of each cDNA is labeled with Digoxigenin-11-dUTP        (Dig-dUTP) (Roche Diagnostics) during PCR. PCR is performed in a        50 μL reaction with 1× Puffer 1, 120 μM CP2 primer, 3% deionized        formamide, 300 μM dTTP, 350 μM dATP, 350 μM dGTP, 350 μM dCTP,        50 μM Dig-dUTP, 4.5 U DNA-Polymerase-Mix. Cycle parameters are:        one cycle: 94° C. for 2 min; 15 cycles: 94° C. for 15 sec,        63° C. for 15 sec, 68° C. for 2 min; 10 cycles: 94° C. for 15        sec, 68° C. for 3 min+5 sec/cycle; one cycle: 68° C., 7 min,        4° C. forever.

Hybridization of Clontech cDNA Arrays with dUTP-labeled cDNA Probes

cDNA arrays are prehybridized in DigEASYHyb solution (Roche Diagnostics)containing 50 mg/L DNAseI (Roche Diagnostics) digested genomic E. coliDNA, 50 mg/L pBluescript plasmid DNA and 15 mg/L herring sperm DNA (LifeTechnologies) for 12 h at 44° C. to reduce background by blockingnon-specific nucleic acid-binding sites on the membrane. Hybridizationsolution is hybridized to commercially available cDNA arrays withselected genes relevant for cancer, cardiovascular and stress response(Clontech). Each cDNA template is denatured and added to theprehybridization solution at a concentration of 5 μg/ml Dig-dUTP-labeledcDNA. Hybridization was performed for 48 hours at 44° C.

-   -   Blots are subsequently rinsed once in 2×SSC/0.1% SDS and once in        1×SSC/0.1% SDS at 68° C. followed by washing for 15 min once in        0.5×SSC/0.1% SDS and twice for 30 min in 0.1×SSC/0.1% SDS at        68° C. For detection of Dig-labeled cDNA hybridized to the        array, the Dig Luminescent Detection Kit (Boehringer, Mannheim)        was used as described in the user manual. For detection of the        chemiluminescence signal, arrays are exposed to        chemiluminescence films for 30 min at room temperature.        Quantification of array data was performed by scanning of the        films and analysis with array vision software (Imaging Research        Inc., St. Catharines, Canada). Background was subtracted and        signals were normalized to the nine housekeeping genes present        on each filter, whereby the average of the housekeeping gene        expression signals was set to 1 and the background set to 0.    -   Each labeled probe was hybridized to three different commercial        cDNA arrays which allowed for the expression analysis of a total        of 2,435 known genes. FIG. 9 shows a representative        hybridization pattern obtained with one array using probes        prepared from restenotic tissue of patient B (panel A) and media        of donor b (panel B). Consistent with the gene-specific analysis        shown in FIG. 8, comparable hybridization signals were obtained        with the positive control of human genomic cDNA spotted on the        right and bottom lanes of the array and with cDNA spots of        various housekeeping genes (see for instance, spots D). If a        biological specimen was omitted from cDNA synthesis and PCR        amplification reactions almost no hybridization signals were        obtained (FIG. 9, panel C), showing that hybridization signals        were almost exclusively derived from added samples and not from        DNA contaminations in reagents or materials used.

Data Analysis

-   -   Quantification of array data was performed by scanning of the        films and analysis with array vision software (Imaging Research        Inc., St. Catharines, Canada). Background was subtracted and        signals were normalized to the nine housekeeping genes present        on each filter, whereby the average of the housekeeping gene        expression signals was set to 1 and the background set to 0. For        the logarithmic presentation shown in FIGS. 13A and 13B, values        were multiplied by 1000. A mean value >0,05 in the average of        all samples in one group was regarded as a positive signal.        Differences in the mean expression level by a factor >2.5-fold        between the study and the control group were further        statistically analyzed.        c) Choice of Control Tissue    -   As the main cellular component of neointima consists of smooth        muscle cells, media and media/intima were taken of healthy        coronary arteries or as coronary arteries belong to the        non-elastic but muscular arteries muscular arteries as control        tissue.    -   The control group consisted of 6 specimen from coronary arteries        from three different patients who underwent heart        transplantation. Additionally, 5 specimen of muscular arteries        of the gastrointestinal tract from five different patients were        taken as control because coronary arteries belong histologically        to muscular arteries. The control specimen were immediately        frozen in liquid nitrogen. Prior to mRNA preparation, media and        intima of the control arteries were prepared and examined for        atherosclerotic changes by immunhistochemistry. If there were no        atherosclerotic changes of the vessel morphology, the specimen        (approx. 1×1 mm) were used as healthy control samples and mRNA        and cDNA preparation and transcriptome analysis was performed as        described above for neointimal tissue.        d) Definition of the Neointima-specific Gene Expression Profile    -   A total of 1,186 genes (48.7%) out of 2,435 yielded detectable        hybridization signals on cDNA arrays with neointima and control        samples over a 20-fold range of expression level (FIG. 13A)        Thereof 352 genes (14.5%) appeared to be differentially        expressed by a factor >2.5-fold between restenotic and control        samples. However, expression levels considerably varied among        individual samples (see, e.g., FIG. 9). A statistical analysis        was therefore employed in order to identify those genes that are        differentially expressed between study and control groups with        high significance (see herein above). This way, 224 genes (9.6%)        were identified that were differentially expressed by a factor        of at least 2.5-fold between the restenosis study group and the        control group with a significance in the Wilcoxon test of        p<0.03. 167 (75%) genes thereof were found overexpressed and 56        genes (25%) underexpressed in the restenosis study group        compared to the control group (FIG. 13B).        ) Choice of Surrogate Cell Line    -   Human neointima consists of a heterogenous cell population. It        was therefore attempted to relate the differential,        statistically relevant gene expression patterns found with        neointima to patterns eventually contributed by peripheral blood        cells of the patients and cultured human CASMCs, i.e., cells        that are most frequently encountered in restenotic        tissue(Komatsu, loc. cit.). With respect to neointima        expression, the 224 aberrantly regulated genes fell into four        subgroups (FIG. 14). Group I lists 62 genes that were        overexpressed in neointima and not highly or detectably        expressed in control vessels, CASMCs or blood cells (FIG. 14A).        In group II, 43 genes are listed that are similarly expressed in        neointima and CASMCs, suggesting that this gene cluster in        neointima was contributed by proliferating SMCs (FIG. 5B). In        group III, 62 genes are listed that are similarly expressed in        neointima and blood cells, suggesting that this gene cluster was        contributed to that of neointima by infiltrated blood cells        (FIG. 14C). This notion is supported by the expression in group        III of the largest number of genes related to inflammation in        all four groups. Lastly, in group IV, 56 genes are listed that        are downregulated in neointima compared to the control group        (FIG. 14D).

Upregulation of γ-IFN-related Genes in Neointima

-   -   A surprising feature of the human neointima transcriptome was        the apparently coordinate upregulation of 32 genes related to        IFN-γ signaling (FIG. 16). The IFN-γ receptor alpha was        expressed in neointima, proliferating CASMCs and—to a lesser        degree—in blood cells; whereas the IFN-γ receptor beta was        mainly expressed in neointima specimen. Consistent with an        activation of IFN-γ signaling, upregulation of two transcription        factors in neointima was found that are essential for IFN        signalling: IRF-1 and ISGF3γ (p48) (FIGS. 14, 15, 16). These        transcription factors are known to be transcriptionally        upregulated by IFN-γ, and both are key players in IFN-γ        signalling. Likewise, upregulation of the tyrosine kinase was        observed Pyk2 (FIG. 16), which has been shown to play a role in        the signal transduction by angiotensin in SMCs (Sabri, Circ.        Res. 83 (1998), 841–851). Pyk2 is selectively activated by IFN-γ        and inhibition of Pyk2 in NIH 3T3 cells results in a strong        inhibition of the IFN-γ-induced activation of MAPK and STAT1. A        key event in IFN-γ-induced growth inhibition and apoptosis is        the induction of caspases (Dai, Blood 93 (1999), 3309–3316). In        the here presented analysis on upregulation of the        IFN-γ-regulated genes for caspase-1, caspase-8 and DAP-1 in        human neointima. However, mRNAs for the the anti-apoptotic        proteins BAG-1, Pim-1 (both regulated by IFN-γ) and        BCL-2-related protein A1 were also upregulated in neointima        versus control (FIG. 16), supporting the notion that        proliferation and apoptosis occur simultaneously in human        neointima with a preponderance of proliferation.    -   Coordinated regulation of genes whose products act at different        steps in the neointima process was a recurring theme of our gene        expression analysis. Regarding the IFN-γ pathway, not only the        genes for the complete receptor, the main transcription factors,        components of the signal transduction pathway (Dap-1, BAG-1,        Pim-1, IFN-γ-inducible protein, IFN-inducible protein 9–27) were        induced but also several target genes of the IFN-γ pathway, like        CD40, CD13 and thrombospondin-1 (FIG. 16).    -   The IFN-γ-regulated gene cluster was expressed in the neointima        specimen but some of the relevant genes, like IRF-1, were also        expressed in blood samples. To identify the cell type that        predominantly contributed to the IFN-γ regulated pattern, frozen        sections of neointima specimen from coronary in-stent restenosis        (n=3) and from restenosis of peripheral arteries (n=6) were        stained with antibodies specific for IRF-1. This protein was        chosen because it is an essential component of the IFN-γ signal        transduction pathway (Kimura, Genes Cells 1 (1996), 115–124) and        was expressed coordinately with the other genes in the cluster        (FIG. 17). Immunohistochemical analysis showed strong nuclear        and cytoplasmic staining of IRF-1 in neointimal SMCs of a        carotid restenosis (FIG. 17B) and of coronary in-stent        restenosis (FIG. 18C), as identified by their spindle-shaped        nuclei and by staining with the smooth muscle cell marker        alpha-actin (FIG. 18B). The nuclear staining of IRF-1 in        in-stent restenosis (FIG. 18C) indicated that the IRF-1        transcription factor is also activated. SMCs in control media of        carotid arteries did not show IRF-1 staining (FIG. 17B).        CD3-positive cells were much less abundant in the specimen (FIG.        18C) than SMCs (FIG. 18D), indicating that SMCs contributed        mostly to the increased IRF-1 expression in human neointima.

Definition of Culturing Conditions in Order to Adapt TranscriptomeProfile to that of Restenotic Tissue: IFN-γ

-   -   To adapt the transcriptional profile of cultured human coronary        artery smooth muscle cells (CASMC) (Clonetics) to that of        neointima, CASMC were stimulated with IFN-g and performed        transcriptome analysis as described above. CASMC were cultured        as described in the manufacter's manual in growth medium until        50% confluency was reached. Afterwards cells were stimulated        with 1000 U/ml IFN-γ (R&D, Germany) for 16 hours at 37° C. Cells        were washed twice in PBS and RNA preparation, cDNA synthesis and        amplification and transcriptome analysis was performed as        described above.    -   As shown in FIG. 19 the neointima-specific IFN-γ gene expression        pattern could be generated by incubation of CASMCs with 1000        U/ml IFN-γ.

Definition of the Transcriptome/gene Expression Pattern of Neointimaafter Incubation with an IFN-γ Antagonist

-   -   Microscopic specimen of in-stent restenotic tissue were        incubated with an antagonist for IFN-γ for different times and        transcriptome analysis was performed as described. Transcriptome        of treated neointima was compared to the transcriptome of        untreated neointima and healthy control tissue, to measured the        therapeutic effect of IFN-γ antagonists.

Definition of th Transcriptom/gene Expression Pattern of Neointima afterIncubation with Rapamycin

-   -   It has been shown in the literature, that rapamycin, a ligand of        the intracellular protein FKBP12 inhibits migration and        proliferation of smooth muscle cells and is able to reduce        neointimal hyperplasia in a porcine model of restenosis. As        significant upregulation of FKBP12 in the neointima specific        transcriptome was found in order to evaluated the therapeutic        effect of rapamycin.    -   As proliferating CASMC overexpress FKBP12 like neointima, this        cell line can be employed as a potential surrogate cell line for        neointima in respect to therapeutic effects of rapamycin.        Therefore, in a first step, CASMC were incubated with 100 ng/ml        rapamycin (Sigma) for 24 hours and transcriptome analysis was        performed in order to monitore the therapeutic effect.        Afterwards, microscopic specimen of in-stent restenotic tissue        are incubated with rapamycin and transcriptome analysis was        performed as described herein above. Transcriptome/gene        expression pattern of rapamycin treated CASMC was compared to        the transcriptome of rapamycin-treated neointima to measured the        effectiveness of CASMC as a surrogate cell line for neointima.        Tumorsuppressor genes and proliferation-inhibiting genes have        upregulated in said CASMCs; therefore said CASMCs can be        considered as an true surrogate for neointima.

EXAMPLE IX Upregulated Protein Expression of Emmprin and TransferrinReceptor on Tumor Cells

Transcriptome analysis of single micrometastatic cells derived frompatients with different tumor and disease stages revealed an upregulatedexpression of genes involved in cell cycle regulation, cytoskeletonorganization, adhesion and proteolytic activity. Enhanced mRNAexpression of Emmprin was found by array hybridization in 10 of 26micrometastatic cells from bone marrow of breast and prostate cancerpatients indicating an invasive phenotype of these cells. EMMPRIN(extracellular matrix metalloproteinase inducer, CD147) is a member ofthe immunoglobulin superfamily that is present on the surface of tumorcells and stimulates adjacent fibroblasts to produce matrixmetalloproteinases (MMPs, Guo, J. Biol. Chem. 272 (1997), 24–27 andSameshima, Cancer Lett. 157 (2000), 177–184 and Li, J. Cell Physiol. 186(2001), 371–379). The results were controlled by gene specific PCRrevealing a similar sensitivity compared to array hybridization. Using adifferent Emmprin-specific probe for array hybridization, the Emmprinmessage was even detected in 16/26 (61%) samples. These resultsemphasize the sensitivity of the array design to detect the transcriptsof a random primed single cell cDNA.

In order to correlate upregulation of Emmprin expression on tumor cellsnot only on mRNA but also on protein level, slides were prepared frombone marrow cells of cancer patients as described before (Pantel, Lancet347 (1996), 649–653). Slides were blocked using 10% human AB serum inPBS for 20 min. From each sample one million bone marrow cells werescreened for the presence of cytokeratin positive cells which is amarker for epithelial cells. A double staining procedure, employing theEMMPRIN specific antibody MEM 6/2 (Koch, Int. Immunol. 11 (1999),777–86) and a biotin-conjugated A45B/B3 antibody reacting with severalcytokeratin family members was performed. Antibody incubations were asfollows: MEM 6/2, 45 min. 5 μg/ml; Z259 and APAAP complex according tothe manufacturer's instructions (DAKO). Slides were washed 3×3 min. inPBS between all antibody incubations. Before the A45 B/B3-biotin F(ab)₂-fragment was added, an additional blocking step with 10% mouseserum in PBS was performed for 20 min. The A45 B/B3-biotin conjugate (2μg/ml; 45 min.) was detected by streptavidin-Cy3 (1.2 μg/ml; 15 min;Jackson laboratories). After washing, FAST-BLUE (Sigma) was used assubstrate for the alkaline phosphatase (10–30 min). For all slides theprocedure was identically performed with isotype controls. EMMPRIN wasdetected on 82% of 140 cytokeratin-positive tumor cells derived from 68patients with breast, prostate and lung cancer (Tab. 8 and FIG. 20). Inonly two aspirates all detected cytokeratin-positive cells (n=4) werenegative for EMMPRIN.

TABLE 8 EMMPRIN (EMM) protein expression on disseminatedcytokeratin-positive (CK+) tumor cells in bone marrow. Number ofpatients with number of double Number of patients with Total numberCK+/EMM+ positive patients CK+ cells of CK+ cells cells cells 68 11/68(16%) 140 115/140 (82%) 9/11 (82%)

Besides Emmprin also expression of transferrin receptor (CD71) on tumorcells was evaluated on protein level. Transcriptome analysis of sixsmall biopsies derived from non-small cell lung cancers and fivebiopsies of control mucosa from patients with chronic obstructivepulmonary showed that signal intensity for CD71 differed greatly betweennormal and tumor tissue (Table 9).

TABLE 9 Signal intensities for the transferrin receptor cDNA on arrayhybridisation Tumor biopsies Normal Mucosa biopsies Bio6 Bio9 Bio10Bio11 Bio1G Bio11G Bio2 Bio3G Bio5G Bio6G Bio14 0 2464 11768 4012 0 54960 0 0 100 0

Differential expression was tested on cryosection of tumor biopsy Bio10and a biopsy from normal mucosa (Bio6G). Unspecific binding was blockedwith 10% AB serum in PBS for 20 minutes and incubation with CD71-PE(phycoerythrin) conjugated antibody (Caltag) was performed for 45minutes. For control an anti-CD4-PE antibody was used. No staining ofthe CD4 antibody was observed on either tissue sample. The CD71-PEantibody selectively stained the epithelial regions of the tumor biopsywhereas the normal mucosa was negative for transferrin receptorexpression (FIG. 21).

EXAMPLE X Anti-apoptotic Effect of IFNγ on Smooth Muscle Cells

The effect of IFNγ on the survival of cultured proliferating SMCs wasanalyzed by flow cytometry. For this reason primary human coronarysmooth muscle cells were obtained from CellSystems (Germany) and weregrown in Smooth muscle cell growth medium (CellSystems) containing 5%fetal calf serum (CellSystems) at 37° C. in a humidified atmosphere of5% CO₂. SMCs were used between passages 2 and 4. Treatment with 1000U/mlrh-IFNγ (R&D Systems) was performed for 16 h. For induction of celldeath, SMCs were incubated at 37° C. for 1 h in HBSS containing 100μmol/l H₂O₂ and 100 □mmol/l ferrous sulfate. Afterwards the cells werefurther cultured in freshly prepared culture medium for 8 h. Cells werelabelled with FITC-labelled Annexin V (Roche Diagnostics) and propidiumiodide (PI) according to the manufacturer's instructions. 10⁴ eventswere analyzed with a flow cytometer (Becton Dickinson).

Flow cytometric analysis revealed an anti-apoptotic effect of IFNγ onSMCs (FIG. 22). FACS analysis after double staining with PI andFITC-labeled Annexin V and showed a reduction of spontaneous apoptosisfrom 10% to 6% after treatment with IFNγ. The effect became moreprominent after induction of apoptosis in SMCs with H₂O₂. Treatment withIFNγ reduced the number of apoptotic cells from 54% to 27%. Theseresults clearly show that IFNγ exerts an anti-apoptotic effect on SMCs.

EXAMPLE XI Inhibitory Effect of IFNγ on Neointima Formation in a MouseModel for Restenosis

To examine the vascular proliferative remodeling after carotid ligation,the mouse blood flow cessation model (Kumar, Circulation 96 (1997),4333–4342) was used. This model is characterized by vascularproliferation of SMCs in response to ligation of the common carotidartery near bifurcation. In order to investigate the effect of an IFN-γreceptor null mutation on the development of neointima in a mouse modelof restenosis IFN-γR^(−/−) knockout mice were used. Adult male 129/svJmice (N=16) and IFN-γR^(−/−) mice (n=11) were anaesthetized byintraperitoneal injection of a solution of xylazine (5 mg/kg bodyweight) and ketamine (80 mg/kg body weight) and the left common carotidartery was ligated near bifurcation. After 4 week animals werereanaesthetized, sacrificied and fixed for 3 min by perfusion with 4%paraformaldehyde in 0.1 mol/l sodium phosphate buffer (pH 7.3,). Afterexcision of the left carotid arteries, vessels were fixed by immersionin 70% ethanol. Carotid arteries were embedded in paraffin and serialsections (% μm thick) were cut.

Morphometric analysis was performed on v.-Giesson stained cross sectionsat a distance of 600 μm from the ligation site. Digitized images of thevessels were analyzed using the image analysis software SCION image4.0.2. Media thickness was obtained as the differences in diameterbetween the external and internal elastic lamina, and neointimathickness as the difference between internal elastic lamina and lumendiameter. Data from morphometric analyses are reported as mean±SEM forthe two groups of mice and tested by the t-test for unpaired samples. Ap value <0.05 was regarded as significant. All analyses were performedwith the use of the SPSS statistical package (version 8.0).

Substantial wall thickening due to media proliferation and neointimaformation was observed in 16 wild-type mice at 4 weeks after ligation(FIG. 23). In 11 IFN-γR^(−/−) mice medial plus neointimal thickening wassignificantly reduced shown as mean±SEM and analyzed by the t-test forunpaired samples. Corresponding to the reduction in proliferativeresponses, 11 IFN-γR^(−/−) mice had a significantly larger lumendiameter of the treated carotid segment than wild-type mice (108±15 umversus 91±24 um and p=0.033).

EXAMPLE XII Suppression Subtractive Hybridization (SSH) Analysis

SSH is a new and highly effective method for the generation ofsubtracted cDNA libraries. Subtractive cDNA hybridization has been apowerful approach to identify and isolate cDNAs of differentiallyexpressed genes (Duguin Nucl. Acid. Res. 18 (1990), 2789–2792 and HaraNucl. Acid. Res. 19 (1991), 7097–7104 and Hendrick Nature (London) 308(1984), 149–153). In general, hybridization of cDNA from one population(tester) to an excess of mRNA (cDNA) from another population (driver)and subsequent separation of the unhybridized fraction (target) from thehybridized common sequences are performed. SSH is used to selectivelyamplify target cDNA fragments (differentially expressed) andsimultaneously suppress nontarget DNA amplification. The method is basedon suppression PCR: long inverted terminal repeats when attached to DNAfragments can selectively suppress amplification of undesirablesequences in PCR procedure. The problem of differences in mRNA abundanceis overcome by a hybridization step that equalizes sequence abundanceduring the course of subtraction. One subtractive hybridization round isrequired leading to a 1000 fold enrichment for differentially expressedcDNAs (for review see Diatchenko Proc. Natl. Acad. Sci. USA 93 (1996),6025–30 and Diatchenko Methods Enzymol. 303 (1999), 349–80).

Serveral modifications were introduced into the standard SSH protocolfor differential gene expression analysis of a very small number ofcells (FIG. 24). 1) mRNA amplificates generated according to the methoddescribed in this patent application had been reverse-transcribed andamplified using CP2 primers; 2) mRNA amplificates generated according tothe method described in this patent application themselves form panlikestructures; 3) introduction of a restriction enzyme recognition site(e.g. EcoRI) into the CP2 primer.

a) Materials and Methods

Oligonucleotides cDNA synthesis primer: CP2: 5′-TCA GAA TTC ATG CCC CCCCCC CCC CCC C-3′ (SEQ ID NO: 14) Adapters Adapter 1 (A1) Eco 44 I:5′-GTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CTC GCC CGG GCA GG-3′ (SEQ IDNO: 31) Eco 12 I: 5′-AAT TCC TGC CCG-3′ (SEQ ID NO: 32) Adapter 2 (A2)Eco 43 II: 5′-TGT AGC GTG AAG ACG ACA GAA AGG TCG CGT GGT GCG GAG GGCG-3′ (SEQ ID NO: 33) Eco 12 II: 5′-AAT TCG CCC TCC-3′ (SEQ ID NO: 34)PCR Primers: P1-30: 5′-GTA ATA CGA CTC ACT ATA GGG CTC GAG CGG-3′ (SEQID NO: 35) P2-30: 5′-TGT AGC GTG AAG ACG ACA GAA AGG TCG CGT-3′ (SEQ IDNO: 36) P1-33: 5′-GTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CTC-3′ (SEQ IDNO: 37) P2-33: 5′-TGT AGC GTG AAG ACG ACA GAA AGG TCG CGT GGT-3′ (SEQ IDNO: 38) PN1-30: 5′-CGA CTC ACT ATA GGG CTC GAG CGG CTC GCC-3′ (SEQ IDNO: 39) PN2-30: 5′-GTG AAG ACG ACA GAA AGG TCG CGT GGT GCG-3′ (SEQ IDNO: 40)

Driver Preparation

For detection of transcripts differentially expressed in micrometastatictumor cells compared to normal bone marrow cells, driver was preparedfrom bone marrow samples derived from healthy donors. From three bonemarrow donors, total RNA was isolated using standard protocols. RNAcorresponding to 300.000 bone marrow cells was then added to 30 μl Dynalbeads and the protocol of mRNA amplification was performed.

Hybridization kinetics were improved by digestion of 5 μg driver with 50units of restriction enzyme Rsa I in a 50 μl reaction containing 0,75×buffer NEB1 (New England Biolabs) for 90 min. The sample was desaltedwith a Microcon 10 column (Millipore).

Tester Preparation

Eco RI digested tester was prepared in 50 μl using 50 U EcoRI. As testera mixture of four single cells isolated from four different breastcancer patients was selected. After digestion with EcoRI the tester wasdiluted to a 100 ng/μl concentration in water. Subsequently, one probewas ligated to 5 μl of adapter A1 (SEQ ID NO: 31, SEQ ID NO: 32) and oneto adapter A2 (SEQ ID NO: 33, SEQ ID NO: 34) (50 μM) in two independent10 μl ligation reactions at 15° C. overnight, using 5 units of T4 DNAligase (Roche). The ligation reaction was inactivated by addition of 2μl 0.1 M EDTA and heating 5 min at 70° C.

Subtractive Hybridization

1 μl of driver (500 ng) was added to each of two tubes containing 2 μlof tester cDNA (about 18 ng) ligated to adapter A1 (SEQ ID NO: 31, SEQID NO: 32) and ligated to adapter A2 (SEQ ID NO: 33, SEQ ID NO: 34) inhybridization buffer (1 M NaCl, 50 mM Hepes, 1 mM CTAB). The solutionwas overlaid with mineral oil, denatured 1 min at 98° C. and thenallowed to anneal for 10–14 hours at 68° C.

After the first hybridization, both samples were mixed together andabout 150 ng heat-denatured driver in 1.5 μl hybridization buffer wereadded. The sample is allowed to hybridize for 10–14 hours. Hybridizationwas stopped by adding 200 μl of dilution buffer (20 mM Hepes, pH 8.3, 50mM NaCl, 0.2 mM EDTA) and by heating for 7 min at 68° C.

PCR Amplification

Two PCR amplification reactions were carried out for each subtraction ina volume of 25 μl. First PCR was performed in Taq long template buffer 1(Roche) with 1 μl of diluted, subtracted cDNA, 1 μl PCR primer P1–30(SEQ ID NO: 35) (8 μM) and 1 μl primer P2–30 (SEQ ID NO: 36) (8 μM) and0.4 mM dNTPs. Taq polymerase was added in a hot start procedure. ThePCR-cycler was set to 75° C. for 7 min (filling in the ends), 27 cycleswere performed (94° C., 30 sec; 66° C., 30 sec; 72° C., 1.5 min) and afinal extension at 72° C. for 7 min. PCR products were diluted 10 foldin water and 1 μl was used for a secondary PCR performed according tothe protocol described above, but using PCR primers PN1–30 (SEQ ID NO:39) and PN2–30 (SEQ ID NO: 40) and 12 cycles (94° C., 30 sec.; 68° C.,30 sec; 72° C., 1.5 min). PCR products were analyzed by gelelectrophoresis on a 1.5% agarose gel.

Cloning and Analysis of Subtracted cDNA

Products from secondary PCR were ligated into the pGEM-Teasy, a T/Acloning system (Promega). After selection of clones withX-Gal/IPTG/ampicilline, inserts were screened by PCR using PN1-30 (SEQID NO: 39) and PN2-30 (SEQ ID NO: 40) primers. Differential expressionwas verified by southern blot analysis of the amplified inserts usinglabeled tester and driver as probes. Labeling of the driver and testersamples was identical to the labeling for array analysis.

Differentially hybridizing clones were subjected to plasmid preparationusing the QIAprep Spin Miniprep Kit (Qiagen) and sequenced. Nucleic acidhomology search was performed using the BLAST program (NCBI).

Results

PCR amplification was performed with primer sets of different length (30nucleotides: P1–30 (SEQ ID NO: 35), P2–30 (SEQ ID NO: 36) and 33nucleotides: P1–33 (SEQ ID NO: 37) and P2–33 (SEQ ID NO: 38)) bothleading to comparable results. Most preferable were primers consistingof 30 nucleotides (P1–30 (SEQ ID NO: 35) and P2–30 (SEQ ID NO: 36)).Smaller primers with 22 nucleotides (Clonetech) as described byDiatchenko (Proc. Natl. Acad. Sci. USA 93 (1996) did not work in PCRreaction. After subtraction, colonies were screened by PCR and theproducts were subjected to gel electrophoresis and blotting. Labeledtester and driver were hybridized onto the blot as shown for one examplein FIG. 25. Colony #4 was identified as a transcription factor describedas epithelium-specific gene (Oettgen Genomics 445, (1997) 456–457 andOettgen Mol. Cell. Biol. 17 (1997), 4419–4433) and Oettgen Genomics 55(1999), 358–62. This result was confirmed by PCR using the samples fromwhich driver and tester had been prepared (FIG. 26).

1. A method for the amplification of mRNA of a sample comprising: i.generating cDNA from polyadenylated RNA employing at least one primerhybridizing to said polyadenylated RNA and comprising a 5′ poly(C) or a5′ poly(G) flank wherein the concentration of said at least one primeris in the range of 10 μM to 60 μM; ii. 3′ tailing of said generated cDNAwith a poly(G) tail when at least one of said primers comprises a 5′poly(C) flank or with a poly(C) tail when at least one of said primerscomprises a 5′ poly(G) flank; and iii. amplifying the tailed cDNA with aprimer hybridizing to the tail(s) generated in ii. to obtain amplifiedcDNA; wherein said at least one primer in step i. comprises SEQ ID NO:10.
 2. The method of claim 1, wherein said primer in step iii comprisesa stretch of at least 10 nucleotides capable of hybridizing with thetail(s) generated in step ii.
 3. The method of claim 2, wherein saidprimer in step iii comprises the sequence selected from the groupconsisting of SEQ ID NOS: SEQ ID NO: 11, 12, 13, 14 and
 15. 4. Themethod of claim 1, wherein said polyadenylated RNA is bound to a solidsupport.
 5. The method of claim 4, wherein said solid support is a bead,a membrane, a filter, a well, a chip or a tube.
 6. The method of claim5, wherein said bead is a magnetic bead, a latex bead or a colloid metalbead.
 7. The method of claim 5, wherein said bead comprises an oligo(dT)stretch.
 8. The method of claim 1, wherein said mRNA is derived from atissue, a low number of cells or a single cell.
 9. The method of claim8, wherein said low number of cells is in a range of 10⁶ to 2 cells. 10.The method of claim 8, wherein said tissue, cells or single cell is ofplant or animal origin.
 11. The method of claim 10, wherein said animalis human.
 12. The method of claim 8, wherein said tissue, low number ofcells or single cell is a chemically fixed tissue, chemically fixed lownumber of cells or chemically fixed cell.
 13. The method of claim 8,wherein said tissue, low number of cells or single cell is derived froma body fluid or from solid tissue.
 14. The method of claim 1, furthercomprising: iv. modifying the generated amplified cDNA of iii.
 15. Themethod of claim 14, wherein said modification comprises the introductionof means for detection.
 16. The method of claim 15, wherein said meansof detection comprises the introduction of nucleotide analogues coupledto (a) chromophore(s), (a) fluorescent dye(s), (a) radio-nucleotide(s),biotin or DIG.
 17. The method of claim 1, wherein the obtained amplifiedcDNA is bound to a solid support.
 18. The method of claim 1, wherein allor individual steps are carried out in a non-cacodylate buffer.
 19. Themethod of claim 18, wherein said non-cacodylate buffer is a phosphatebuffer.
 20. The method of claim 19, wherein said phosphate-buffer is aKH₂PO₄ buffer.
 21. The method claim 1, wherein said sample is derivedfrom a cell and/or a tissue, the genetic identity of which had beendefined by comparative genomic hybridization.
 22. A method for thepreparation of an in vitro surrogate for at least one pathologicallymodified cell or tissue comprising: (a) amplifying mRNA of said at leastone pathologically modified cell or tissue according to the method ofclaim 1; (b) assessing the quantity and, optionally, biophysicalcharacteristics of the amplified cDNA and/or transcripts thereof,thereby determining the gene expression pattern of said pathologicallymodified cell(s) or tissue(s); (c) selecting a human CASMC cell treatedwith IFN-γ, the gene expression pattern of which resembles the geneexpression pattern of said at least one pathologically modified cell ortissue; and (d) adapting the gene expression pattern of said CASMC cellto the gene expression pattern of the pathologically modified cell ortissue, wherein said pathologically modified cell or tissue is a humanrestenotic cell or restenotic tissue.
 23. The method of claim 22,further comprising in (b): i. determining the gene expression pattern ofat least one control cell or control tissue; and ii. determining atleast one gene which is differentially expressed in said pathologicallymodified cell or tissue and said control cell or tissue.
 24. The methodof claim 23, wherein said gene expression pattern of a control cell or acontrol tissue is determined employing the method of RNA amplificationused to amplify the pathologically modified cell or tissue.
 25. Themethod of claim 23, wherein said control cells or tissue is smoothmuscle cells or media/intima of healthy coronary arteries.
 26. Themethod of claim 22, wherein said in vitro cell is or is derived form aprimary cell culture, a secondary cell culture, a tissue culture or acell line.
 27. The method of claim 26, wherein said in vitro cell isselected from the group consisting of cultured coronary artery smoothmuscle cells, HepG2 cells, Jurkat cells, THP-I cells, Monomac-6-cells,U937 cells, ATCC 45505 cells, cultured cardiomyocytes, ECV 304 cells andNIH3T3 cells.
 28. The method of claim 22, wherein said adaptation instep c comprises the exposure of said in vitro cell to physical and/orchemical changes.
 29. The method of claim 28, wherein said physicalchanges comprise temperature shifts, light changes, pH-changes in ionicstrength or changes in the gas phase.
 30. The method of claim 28,wherein said chemical changes comprise medium exchanges, mediumsubstitutions, medium depletions and/or medium additions.
 31. The methodof claim 28, wherein said chemical changes comprise the exposure tocompounds selected from the group consisting of growth factors,hormones, vitamins, antibodies or fragments and/or derivatives thereof,cytokines, transcription factors, kinases, antibiotics, natural receptorligands, non-natural receptor ligands and components of signaltransduction pathways.
 32. The method of claim 31, wherein said cytokineis IFN-γ or a functional derivative thereof, said natural or non-naturalreceptor ligand is a ligand for IFN-γ receptor, said transcriptionfactor is IRF-1 or ISGF3-γ-(p48), said kinase is tyrosine kinase Pyk2,said components of signal transduction pathways is Dap-1, BAG-1, Pim-1or IFN-γ-inducible protein 9–27, said growth factor is platelet growthfactor AA, angiotension or fibroblast growth factor or said antibioticis rapamycin.
 33. A method for identifying differentially expressedgenes in a test sample comprising: (a) providing a test sample and acontrol sample each comprising polyadenylated RNA; (b) employing thesteps of the method of claim 1 on said test and control sample; and (c)comparing the obtained amplified cDNA of said test sample with theobtained amplified cDNA of said control sample.
 34. A method foridentifying a drug candidate for prevention or therapy of a pathologicalcondition or a pathological disorder comprising: (a) contacting a samplecomprising polyadenylated RNA with a drug candidate; (b) employing thesteps of the method of claim 1 on said sample; and (c) detecting thepresence, the absence, the increase or the decrease of particularexpressed genes in said sample, wherein the correlation of saidpresence, absence, increase or decrease with the presence of said drugcandidate qualifies said drug candidate as a drug.
 35. A method for invitro detection of a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) providing a samplecomprising polyadenylated RNA from said subject; (b) employing the stepsof the method of claim 1 on said sample; and (c) detecting apathological condition or a susceptibility to a pathological conditionbased on the presence, the absence, the increase, the decrease or theamount of (a) expressed gene(s) in said sample.
 36. A method ofidentifying a drug candidate for prevention or therapy of a pathologicalcondition or a pathological disorder in an in vitro surrogatecomprising: (a) contacting an in vitro surrogate for at least onepathologically modified cell or tissue produced according to the methodof claim 22 with a drug candidate; and (b) detecting the presence, theabsence, the increase or the decrease of particular expressed genes insaid sample, wherein the correlation of said presence, absence, increaseor decrease with the presence of said drug candidate qualifies said drugcandidate as a drug, wherein said pathological condition or pathologicaldisorder is restenosis and said pathologically modified cell or tissueis a human restenotic cell or restenotic tissue.
 37. A method ofutilizing amplified cDNA comprising: (a) preparing amplified cDNAaccording to the method of claim 1, and (b) utilizing said amplifiedcDNA in a method selected from the group consisting of in vitroexpression of proteins or polypeptides, in vivo expression of proteinsor polypeptides, in vitro preparation of mRNA transcripts, in vivopreparation of mRNA transcripts, hybridization assays, interactionassays, sequence specific PCR, cDNA cloning, substractive hybridizationcloning and expression cloning.
 38. The method of claim 37, wherein saidhybridization assay comprises the hybridization to oligonucleotidearrays, cDNA arrays, and/or PNA arrays.
 39. The method of claim 37,wherein said interaction assay comprises the interaction with at leastone carbohydrate, lectin, ribozyme, protein, peptide, antibody, antibodyfragment, aptamer or a combination thereof.
 40. A kit comprising SEQ IDNO:10.